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N 6 -methylenadenosine (m 6 A) modification, the most abundant epitranscriptomic modification in eukaryotic mRNAs, has been shown to play crucial roles in regulating various aspects of mRNA metabolism and functions. In this study, we applied the Cre-Loxp conditional knockout system to investigate the role of the core components of the m 6 A methyltransferase complex, METTL14 and METTL3, in retinal development. Our results showed that the double absence of Mettl14 and Mettl3 caused structural disturbance in the retina and prolonged the proliferation activity of retinal progenitor cells. Interestingly, the deletion of Mettl14 and Mettl3 did not affect the generation of various retinal cells, but severely disrupted their distribution. In addition, double deletion of Mettl14 together with Mettl3 caused a stronger phenotype than did single deletion of Mettl14. In conclusion, our study demonstrated that Mettl14 and Mettl3 work cooperatively to regulate retinal development., (© 2024 John Wiley & Sons Ltd.)

Background/objective: The Rs1 exon-1-del rat (Rs1KO) XLRS model shows normal retinal development until postnatal day 12 (P12) when small cystic spaces start to form in the inner nuclear layer. These spaces enlarge rapidly, peak at P15, and then collapse by P19., Methods: We explored the possible involvement of Kir4.1 and Aqp4, the principal retina channels for water movement and homeostasis, along with Muller glia cells (MGCs), using semi-quantitative fluorescent immunohistochemistry at P7, P9, P12, and P30, in Rs1KO and WT littermates., Results: Kir4.1 expression was reduced in Rs1KO retinas at all the early time points-P7, P9, and P12-as the schisis cavities began to form; downregulation would reduce water egress from the retina. Aqp4 was upregulated at P30 in Rs1KO retinas during schisis cavity closure but not as cavities formed at P12. When examined by GFAP expression, MGCs were not activated at the preschisis P12 age but showed considerable GFAP expression at P30 following retinal cystic structural damage at P15, indicating that MGCs were activated during the period of retina water removal and cavity closure., Conclusions: The study results implicate the downregulation of Kir4.1 in schisis formation and a role for both Kir4.1 and Aqp4 upregulation in subsequent schisis closure.

Nuclear positioning is a crucial aspect of cell and developmental biology. One example is the apical movement of nuclei in neuroepithelia before mitosis, which is essential for proper tissue formation. While the cytoskeletal mechanisms that drive nuclei to the apical side have been explored, the influence of nuclear properties on apical nuclear migration is less understood. Nuclear properties, such as deformability, can be linked to lamin A/C expression levels, as shown in various in vitro studies. Interestingly, many nuclei in early development, including neuroepithelial nuclei, express only low levels of lamin A/C. Therefore, we investigated whether increased lamin A expression in the densely packed zebrafish retinal neuroepithelium affects nuclear deformability and, consequently, migration phenomena. We found that overexpressing lamin A in retinal nuclei increases nuclear stiffness, which in turn indeed impairs apical nuclear migration. Interestingly, nuclei that do not overexpress lamin A but are embedded in a stiffer lamin A-overexpressing environment also exhibit impaired apical nuclear migration, indicating that these effects can be cell non-autonomous. Additionally, in the less crowded hindbrain neuroepithelium, only minor effects on apical nuclear migration are observed. Together, this suggests that the material properties of the nucleus influence nuclear movements in a tissue-dependent manner., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Jamming of cell collectives and associated rigidity transitions have been shown to play a key role in tissue dynamics, structure and morphogenesis. Cellular jamming is controlled by cellular density and the mechanics of cell-cell contacts. However, the contribution of subcellular organelles to the physical state of the emergent tissue is unclear. Here we report a nuclear jamming transition in zebrafish retina and brain tissues, where physical interactions between highly packed nuclei restrict cellular movements and control tissue mechanics and architecture. Computational modelling suggests that the nuclear volume fraction and anisotropy of cells control the emerging tissue physical state. Analysis of tissue architecture, mechanics and nuclear movements during eye development show that retina tissues undergo a nuclear jamming transition as they form, with increasing nuclear packing leading to more ordered cellular arrangements, reminiscent of the crystalline cellular packings in the functional adult eye. Our results reveal an important role of the cell nucleus in tissue mechanics and architecture., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The development of the visual system is a complex and multistep process characterized by the precise wiring of retinal ganglion cell (RGC) axon terminals with their corresponding neurons in the visual nuclei of the brain. Upon reaching primary image-forming nuclei (IFN), such as the superior colliculus and the lateral geniculate nucleus, RGC axons undergo extensive arborization that refines over the first few postnatal weeks. The molecular mechanisms driving this activity-dependent remodeling process, which is influenced by waves of spontaneous activity in the developing retina, are still not well understood. In this study, by manipulating the activity of RGCs in mice from either sex and analyzing their transcriptomic profiles before eye-opening, we identified the Type I membrane protein synaptotagmin 13 (Syt13) as involved in spontaneous activity-dependent remodeling. Using these mice, we also explored the impact of spontaneous retinal activity on the development of other RGC recipient targets such as nonimage-forming (NIF) nuclei and demonstrated that proper frequency and duration of retinal waves occurring prior to visual experience are essential for shaping the connectivity of the NIF circuit. Together, these findings contribute to a deeper understanding of the molecular and physiological mechanisms governing activity-dependent axon refinement during the assembly of the visual circuit., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

Alternative splicing (AS) is a crucial mechanism contributing to proteomic diversity, which is highly regulated in tissue- and development-specific patterns. Retinal tissue exhibits one of the highest levels of AS. In particular, photoreceptors have a distinctive AS pattern involving the inclusion of microexons not found in other cell types. PROM1 whose encoded protein Prominin-1 is located in photoreceptor outer segments (OSs), undergoes exon 4 inclusion from the 12 th post-conception week of human development through adulthood. Exon 4 skipping in PROM1 is associated with late-onset mild maculopathy, however its role in photoreceptor maturation and function is unknown. In this study retinal organoids, a valuable model system, were employed in combination with phosphorodiamidate morpholino oligos (PMOs) to assess the role of exon 4 AS in the development of human retina. Retinal organoids were treated with the PMOs for four weeks after which RT-PCR, western blotting and immunofluorescence analysis were performed to assess exon 4 exclusion and its impact on photoreceptors. The transcriptome of treated ROs was studied by bulk RNA-Seq. Our data demonstrate that 55% skipping of PROM1 exon 4 resulted in decreased Prominin-1 expression by 40%, abnormal accumulation of cones in the basal side of the retinal organoids as well as detectable cone photoreceptor cilium defects. Transcriptomic and western blot analyses revealed decreased expression of cone, inner segment and connecting cilium basal body markers, increased expression of genes associated with stress response and the ubiquitin-proteasome system, and downregulation of autophagy. Importantly, the use of retinal organoids provides a valuable platform to study AS and unravel disease mechanisms in a more physiologically relevant context, opening avenues for further research and potential therapeutic interventions. Together our data indicate that cones may be more sensitive to PROM1 exon 4 skipping and/or reduced Prominin-1 expression, corroborating the pathogenesis of late-onset mild maculopathy., (© 2024. The Author(s).)

This study aimed to explore how Dab1 functional silencing influences the expression patterns of different connexins in the developing yotari ( yot ) mice eyes as potential determinants of retinogenesis. Using immunofluorescence staining, the protein expression of Dab1, Reelin, and connexin 37, 40, 43, and 45 (Cx37, Cx40, Cx43, and Cx45) in the wild-type (wt) and yot eyes at embryonic days 13.5 and 15.5 (E13.5 and E15.5) were analyzed. Different expression patterns of Cx37 were seen between the wt and yot groups. The highest fluorescence intensity of Cx37 was observed in the yot animals at E15.5. Cx40 had higher expression at the E13.5 when differentiation of retinal layers was still beginning, whereas it decreased at the E15.5 when differentiation was at the advanced stage. Higher expression of Cx43 was found in the yot group at both time points. Cx45 was predominantly expressed at E13.5 in both groups. Our results reveal the altered expression of connexins during retinogenesis in yot mice and their potential involvement in retinal pathology, where they might serve as prospective therapeutic targets.

Retinal ganglion cell (RGC) axons provide direct input into several brain regions, including the dorsal lateral geniculate nucleus (dLGN), which is important for image-forming vision, and the ventrolateral geniculate nucleus (vLGN), which is associated with nonimage-forming vision. Through both activity- and morphogen-dependent mechanisms, retinal inputs play important roles in the development of dLGN, including the refinement of retinal projections, morphological development of thalamocortical relay cells (TRCs), timing of corticogeniculate innervation, and recruitment and distribution of inhibitory interneurons. In contrast, little is known about the role of retinal inputs in the development of vLGN. Grossly, vLGN is divided into two domains, the retinorecipient external vLGN (vLGNe) and nonretinorecipient internal vLGN (vLGNi). Studies previously found that vLGNe consists of transcriptionally distinct GABAergic subtypes distributed into at least four adjacent laminae. At present, it remains unclear whether retinal inputs influence the development of these cell-type-specific neuronal laminae in vLGNe. Here, we elucidated the developmental timeline for these laminae in the mouse vLGNe, and results indicate that these laminae are specified at or before birth. We observed that mutant mice without retinal inputs have a normal laminar distribution of GABAergic cells at birth; however, after the first week of postnatal development, these mutants exhibited a dramatic disruption in the laminar organization of inhibitory neurons and clear boundaries between vLGNe and vLGNi. Overall, our results show that while the formation of cell-type-specific layers in mouse vLGNe does not depend on RGC inputs, retinal signals are critical for their maintenance., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 Stebbins et al.)

Cell shape remodeling is a principal driver of epithelial tissue morphogenesis. While progress continues to be made in our understanding of the pathways that control the apical (top) geometry of epithelial cells, we know comparatively little about those that control cell basal (bottom) geometry. To examine this, we used the Drosophila ommatidium, which is the basic visual unit of the compound eye. The ommatidium is shaped as a hexagonal prism, and generating this 3D structure requires ommatidial cells to adopt specific apical and basal polygonal geometries. Using this model system, we find that generating cell type-specific basal geometries starts with patterning of the basal extracellular matrix, whereby Laminin accumulates at discrete locations across the basal surface of the retina. We find the Dystroglycan receptor complex (DGC) is required for this patterning by promoting localized Laminin accumulation at the basal surface of cells. Moreover, our results reveal that localized accumulation of Laminin and the DGC are required for directing Integrin adhesion. This induces cell basal geometry remodeling by anchoring the basal surface of cells to the extracellular matrix at specific, Laminin-rich locations. We propose that patterning of a basal extracellular matrix by generating discrete Laminin domains can direct Integrin adhesion to induce cell shape remodeling in epithelial morphogenesis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Walther et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The development of the retina is under tight temporal and spatial control. To gain insights into the molecular basis of this process, we generate a single-nuclei dual-omic atlas of the human developing retina with approximately 220,000 nuclei from 14 human embryos and fetuses aged between 8 and 23-weeks post-conception with matched macular and peripheral tissues. This atlas captures all major cell classes in the retina, along with a large proportion of progenitors and cell-type-specific precursors. Cell trajectory analysis reveals a transition from continuous progression in early progenitors to a hierarchical development during the later stages of cell type specification. Both known and unrecorded candidate transcription factors, along with gene regulatory networks that drive the transitions of various cell fates, are identified. Comparisons between the macular and peripheral retinae indicate a largely consistent yet distinct developmental pattern. This atlas offers unparalleled resolution into the transcriptional and chromatin accessibility landscapes during development, providing an invaluable resource for deeper insights into retinal development and associated diseases., (© 2024. The Author(s).)

The non-canonical Wnt pathway is an evolutionarily conserved pathway essential for tissue patterning and development across species and tissues. In mammals, this pathway plays a role in neuronal migration, dendritogenesis, axon growth, and synapse formation. However, its role in development and synaptogenesis of the human retina remains less established. In order to address this knowledge gap, we analyzed publicly available single-cell RNA sequencing (scRNAseq) datasets for mouse retina, human retina, and human retinal organoids over multiple developmental time points during outer retinal maturation. We identified ligands, receptors, and mediator genes with a putative role in retinal development, including those with novel or species-specific expression, and validated this expression using fluorescence in situ hybridization (FISH). By quantifying outer nuclear layer (ONL) versus inner nuclear layer (INL) expression, we provide evidence for the differential expression of certain non-canonical Wnt signaling components in the developing mouse and human retina during outer plexiform layer (OPL) development. Importantly, we identified distinct expression patterns of mouse and human FZD3 and WNT10A, as well as previously undescribed expression, such as for mouse Wnt2b in Chat + starburst amacrine cells. Human retinal organoids largely recapitulated the human non-canonical Wnt pathway expression. Together, this work provides the basis for further study of non-canonical Wnt signaling in mouse and human retinal development and synaptogenesis., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Cell fate progression of pluripotent progenitors is strictly regulated, resulting in high human cell diversity. Epigenetic modifications also orchestrate cell fate restriction. Unveiling the epigenetic mechanisms underlying human cell diversity has been difficult. In this study, we use human brain and retina organoid models and present single-cell profiling of H3K27ac, H3K27me3 and H3K4me3 histone modifications from progenitor to differentiated neural fates to reconstruct the epigenomic trajectories regulating cell identity acquisition. We capture transitions from pluripotency through neuroepithelium to retinal and brain region and cell type specification. Switching of repressive and activating epigenetic modifications can precede and predict cell fate decisions at each stage, providing a temporal census of gene regulatory elements and transcription factors. Removing H3K27me3 at the neuroectoderm stage disrupts fate restriction, resulting in aberrant cell identity acquisition. Our single-cell epigenome-wide map of human neural organoid development serves as a blueprint to explore human cell fate determination., (© 2024. The Author(s).)

Human stem cell-derived organoids enable both disease modeling and serve as a source of cells for transplantation. Human retinal organoids are particularly important as a source of human photoreceptors; however, the long differentiation period required and lack of vascularization in the organoid often results in a necrotic core and death of inner retinal cells before photoreceptors are fully mature. Manipulating the in vitro environment of differentiating retinal organoids through the incorporation of extracellular matrix components could influence retinal development. We investigated the addition of hyaluronan (HA), a component of the interphotoreceptor matrix, as an additive to promote long-term organoid survival and enhance retinal maturation. HA treatment had a significant reduction in the proportion of proliferating (Ki67+) cells and increase in the proportion of photoreceptors (CRX+), suggesting that HA accelerated photoreceptor commitment in vitro. HA significantly upregulated genes specific to photoreceptor maturation and outer segment development. Interestingly, prolonged HA-treatment significantly decreased the length of the brush border layer compared to those in control retinal organoids, where the photoreceptor outer segments reside; however, HA-treated organoids also had more mature outer segments with organized discs structures, as revealed by transmission electron microscopy. The brush border layer length was inversely proportional to the molar mass and viscosity of the hyaluronan added. This is the first study to investigate the role of exogenous HA, viscosity, and polymer molar mass on photoreceptor maturation, emphasizing the importance of material properties on organoid culture. STATEMENT OF SIGNIFICANCE: Retinal organoids are a powerful tool to study retinal development in vitro, though like many other organoid systems, can be highly variable. In this work, Shoichet and colleagues investigated the use of hyaluronan (HA), a native component of the interphotoreceptor matrix, to improve photoreceptor maturation in developing human retinal organoids. HA promoted human photoreceptor differentiation leading to mature outer segments with disc formation and more uniform and healthy retinal organoids. These findings highlight the importance of adding components native to the developing retina to generate more physiologically relevant photoreceptors for cell therapy and in vitro models to drive drug discovery and uncover novel disease mechanisms., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: KK, YU, HN, HN, and YH are employees at Rohto Pharmaceuticals. The authors have a filed a patent on the application of HA on retinal organoids., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In this work we present a detailed study of the major events during retinal histogenesis of the cuttlefish Sepia officinalis from early embryos to newly hatched animals and juveniles. For this purpose, we carried out morphometric and histological analyses using light and scanning electron microscopy. From St19, the first embryonic stage analysed, to St23/24 the embryonic retina is composed of a pseudostratified epithelium showing abundant mitotic figures in the more internal surface. At St24 the first photoreceptor nuclei appear in the presumptive inner segment layer, while an incipient layer of apical processes of the future rhabdomeric layer become visible at St25. From this stage onwards, both the rhabdomeric layer and the inner segment layer increase in size until postnatal ages. In contrast, the width of the supporting cell layer progressively decreases from St25/26 until postnatal ages. S. officinalis embryos hatched in a morphologically advanced state, showing a differentiated retina even in the last stages of the embryonic period. However, features of immaturity are still observable in the retinal tissue during the first postnatal weeks of life, such as the existence of mitotic figures in the apical region of the supporting cell layer and migrating nuclei of differentiating photoreceptors crossing the basal membrane to reach their final location in the inner segment layer. Therefore, postnatal retinal neurogenesis is present in juvenile specimens of S. officinalis., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

While dysfunction and death of light-detecting photoreceptor cells underlie most inherited retinal dystrophies, knowledge of the species-specific details of human rod and cone photoreceptor cell development remains limited. Here, we generated retinal organoids carrying retinal disease-causing variants in NR2E3, as well as isogenic and unrelated controls. Organoids were sampled using single-cell RNA sequencing (scRNA-Seq) across the developmental window encompassing photoreceptor specification, emergence, and maturation. Using scRNA-Seq data, we reconstruct the rod photoreceptor developmental lineage and identify a branch point unique to the disease state. We show that the rod-specific transcription factor NR2E3 is required for the proper expression of genes involved in phototransduction, including rhodopsin, which is absent in divergent rods. NR2E3-null rods additionally misexpress several cone-specific phototransduction genes. Using joint multimodal single-cell sequencing, we further identify putative regulatory sites where rod-specific factors act to steer photoreceptor cell development. Finally, we show that rod-committed photoreceptor cells form and persist throughout life in a patient with NR2E3-associated disease. Importantly, these findings are strikingly different from those observed in Nr2e3 rodent models. Together, these data provide a road map of human photoreceptor development and leverage patient induced pluripotent stem cells to define the specific roles of rod transcription factors in photoreceptor cell emergence and maturation in health and disease.

Haploinsufficiency of the PRR12 gene is implicated in a human neuro-ocular syndrome. Although identified as a nuclear protein highly expressed in the embryonic mouse brain, PRR12 molecular function remains elusive. This study explores the spatio-temporal expression of zebrafish PRR12 co-orthologs, prr12a and prr12b , as a first step to elucidate their function. In silico analysis reveals high evolutionary conservation in the DNA-interacting domains for both orthologs, with significant syntenic conservation observed for the prr12b locus. In situ hybridization and RT-qPCR analyses on zebrafish embryos and larvae reveal distinct expression patterns: prr12a is expressed early in zygotic development, mainly in the central nervous system, while prr12b expression initiates during gastrulation, localizing later to dopaminergic telencephalic and diencephalic cell clusters. Both transcripts are enriched in the ganglion cell and inner neural layers of the 72 hpf retina, with prr12b widely distributed in the ciliary marginal zone. In the adult brain, prr12a and prr12b are found in the cerebellum, amygdala and ventral telencephalon, which represent the main areas affected in autistic patients. Overall, this study suggests PRR12 's potential involvement in eye and brain development, laying the groundwork for further investigations into PRR12 -related neurobehavioral disorders.

Background: By examining species-specific innate behaviours, neuroethologists have characterized unique neural strategies and specializations from throughout the animal kingdom. Simultaneously, the field of evolutionary developmental biology (informally, "evo-devo") seeks to make inferences about animals' evolutionary histories through careful comparison of developmental processes between species, because evolution is the evolution of development. Yet despite the shared focus on cross-species comparisons, there is surprisingly little crosstalk between these two fields. Insights can be gleaned at the intersection of neuroethology and evo-devo. Every animal develops within an environment, wherein ecological pressures advantage some behaviours and disadvantage others. These pressures are reflected in the neurodevelopmental strategies employed by different animals across taxa., Summary: Vision is a system of particular interest for studying the adaptation of animals to their environments. The visual system enables a wide variety of animals across the vertebrate lineage to interact with their environments, presenting a fantastic opportunity to examine how ecological pressures have shaped animals' behaviours and developmental strategies. Applying a neuroethological lens to the study of visual development, we advance a novel theory that accounts for the evolution of spontaneous retinal waves, an important phenomenon in the development of the visual system, across the vertebrate lineage., Key Messages: We synthesize literature on spontaneous retinal waves from across the vertebrate lineage. We find that ethological considerations explain some cross-species differences in the dynamics of retinal waves. In zebrafish, retinal waves may be more important for the development of the retina itself, rather than the retinofugal projections. We additionally suggest empirical tests to determine whether Xenopus laevis experiences retinal waves., (© 2024 The Author(s). Published by S. Karger AG, Basel.)

Müller glial cells, which are the most predominant glial subtype in the retina, play multiple important roles, including the maintenance of structural integrity, homeostasis, and physiological functions of the retina. We have previously found that the Rax homeoprotein is expressed in postnatal and mature Müller glial cells in the mouse retina. However, the function of Rax in postnatal and mature Müller glial cells remains to be elucidated. In the current study, we first investigated Rax function in retinal development using retroviral lineage analysis and found that Rax controls the specification of late-born retinal cell types, including Müller glial cells in the postnatal retina. We next generated Rax tamoxifen-induced conditional KO (Rax iCKO) mice, where Rax can be depleted in mTFP-labeled Müller glial cells upon tamoxifen treatment, by crossing Rax flox/flox mice with Rlbp1-CreERT2 mice, which we have produced. Immunohistochemical analysis showed a characteristic of reactive gliosis and enhanced gliosis of Müller glial cells in Rax iCKO retinas under normal and stress conditions, respectively. We performed RNA-seq analysis on mTFP-positive cells purified from the Rax iCKO retina and found significantly reduced expression of suppressor of cytokinesignaling-3 (Socs3). Reporter gene assays showed that Rax directly transactivates the Socs3 promoter. We observed decreased expression of Socs3 in Müller glial cells of Rax iCKO retinas by immunostaining. Taken together, the present results suggest that Rax suppresses inflammation in Müller glial cells by transactivating Socs3. This study sheds light on the transcriptional regulatory mechanisms underlying retinal Müller glial cell homeostasis., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Mice have emerged as a widely employed model for investigating various retinal diseases. However, the availability of comprehensive datasets capturing the entire developmental and aging stages of the mouse retina, particularly during the elderly period, encompassing integrated lncRNA and mRNA expression profiles, is limited. In this study, we assembled a total of 18 retina samples from mice across 6 distinct stages of development and aging (5 days, 3 weeks, 6 weeks, 10 weeks, 6 months, and 15 months) to conduct integrated lncRNA and mRNA sequencing analysis. This invaluable dataset offers a comprehensive transcriptomic resource of mRNA and lncRNA expression profiles during the natural progression of retinal development and aging. The discoveries stemming from this investigation will significantly contribute to the elucidation of the underlying molecular mechanisms associated with various retinal diseases, such as congenital retinal dysplasia and retinal degenerative diseases., (© 2023. Springer Nature Limited.)

Drosophila compound eye morphogenesis transforms a simple epithelium into an approximate hollow hemisphere comprised of ∼700 ommatidia, packed as tapering hexagonal prisms between a rigid external array of cuticular lenses and a parallel, rigid internal floor, the fenestrated membrane (FM). Critical to vision, photosensory rhabdomeres are sprung between these two surfaces, grading their length and shape accurately across the eye and aligning them to the optical axis. Using fluorescently tagged collagen and laminin, we show that that the FM assembles sequentially, emerging in the larval eye disc in the wake of the morphogenetic furrow as the original collagen-containing basement membrane (BM) separates from the epithelial floor and is replaced by a new, laminin-rich BM, which advances around axon bundles of newly differentiated photoreceptors as they exit the retina, forming fenestrae in this new, laminin-rich BM. In mid-pupal development, the interommatidial cells (IOCs) autonomously deposit collagen at fenestrae, forming rigid, tension-resisting grommets. In turn, stress fibers assemble in the IOC basal endfeet, where they contact grommets at anchorages mediated by integrin linked kinase (ILK). The hexagonal network of IOC endfeet tiling the retinal floor couples nearest-neighbor grommets into a supracellular tri-axial tension network. Late in pupal development, IOC stress fiber contraction folds pliable BM into a hexagonal grid of collagen-stiffened ridges, concomitantly decreasing the area of convex FM and applying essential morphogenetic longitudinal tension to rapidly growing rhabdomeres. Together, our results reveal an orderly program of sequential assembly and activation of a supramolecular tensile network that governs Drosophila retinal morphogenesis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Myelination allows for the regulation of conduction velocity, affecting the precise timing of neuronal inputs important for the development and function of brain circuits. In turn, myelination may be altered by changes in experience, neuronal activity, and vesicular release, but the links between sensory experience, corresponding neuronal activity, and resulting alterations in myelination require further investigation. We thus studied the development of myelination in the Xenopus laevis tadpole, a classic model for studies of visual system development and function because it is translucent and visually responsive throughout the formation of its retinotectal system. We begin with a systematic characterization of the timecourse of early myelin ensheathment in the Xenopus retinotectal system using immunohistochemistry of myelin basic protein (MBP) along with third harmonic generation (THG) microscopy, a label-free structural imaging technique. Based on the mid-larval developmental progression of MBP expression in Xenopus, we identified an appropriate developmental window in which to assess the effects of early temporally patterned visual experience on myelin ensheathment. We used calcium imaging of axon terminals in vivo to characterize the responses of retinal ganglion cells over a range of stroboscopic stimulation frequencies. Strobe frequencies that reliably elicited robust versus dampened calcium responses were then presented to animals for 7 d, and differences in the amount of early myelin ensheathment at the optic chiasm were subsequently quantified. This study provides evidence that it is not just the presence but also to the specific temporal properties of sensory stimuli that are important for myelin plasticity., (© 2021 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals LLC.)

The retina is a complex system containing several neuron types arranged in distinct layers. Many aspects of the retina's development and the molecular events in the human light-sensing system have been previously unveiled. However, there is limited information about regulatory networks governing the transitional stages during retina development. To address this issue, we have studied the transcriptome dynamics of mice-derived retinal organoid development in 10 successive time-points, from stem cell to functional retina. For the first time, we have identified the main modules of genes related to different stages of development and predicted all possible transcription factors. A major shift in the transcriptome occurs during the transition of cells from D0 to D10 and again at the late stages of retina development. Transcription, nervous system development, cell cycle, neurotransmitter transport, glycosylation, and lipid metabolisms are the most important biological processes during retina development. Altogether, we have identified and reported 15 TFs, including Irx2, Irx3, Lmo2, Tead2, Tbx20, and Zeb1, which are potentially involved in the regulation of retinal organoid development. In conclusion, using several rigorous analyses, we have found main stage-specific biological processes in the retina development and predicted TFs with strong potency in controlling this structure., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Aim: Eye organoids are 3D models of the retina that provide new possibilities for studying retinal development, drug toxicity and the molecular mechanisms of diseases. Although there are several protocols that can be used to generate functional tissues, none have been used to assemble human retinal organoids containing mesenchymal stem cells (MSCs)., Main Methods: In this study we intend to assess the effective interactions of MSCs and human embryonic stem cells (hESCs) during retinal organoid formation. We evaluated the inducing activities of bone marrow MSCs (BM-MSCs), trabecular meshwork (TM), and stem cells from apical papilla (SCAP)-derived MSCs in differentiation of hESCs in a three-dimensional (3D) direct co-culture system., Key Findings: In comparison with the two other MSC sources, the induction potential of SCAP was confirmed in the co-culture system. Although the different SCAP cell ratios did not show any significant morphology changes during the first seven days, increasing the number of SCAPs improved formation of the optic vesicle (OV) structure, which was confirmed by assessment of specific markers. The OVs subsequently developed to an optic cup (OC), which was similar to the in vivo environment. These arrangements expressed MITF in the outer layer and CHX10 in the inner layer., Significance: We assessed the inducing activity of SCAP during differentiation of hESCs towards a retinal fate in a 3D organoid system. However, future studies be conducted to gather additional details about the development of the eye field, retinal differentiation, and the molecular mechanisms of diseases., (Copyright © 2021. Published by Elsevier Inc.)

The Zic family of zinc finger transcription factors plays a critical role in multiple developmental processes. Using loss-of-function studies, we find that Zic5 is important for the differentiation of retinal pigmented epithelium (RPE) and the rod photoreceptor layer through suppressing Hedgehog (Hh) signaling. Further, Zic5 interacts with the critical Hh signaling molecule, Gli3, through the zinc finger domains of both proteins. This Zic5-Gli3 interaction disrupts Gli3/Gli3 homodimerization, resulting in Gli3 protein stabilization via a reduction in Gli3 ubiquitination. During embryonic Hh signaling, the activator form of Gli is normally converted to a repressor form through proteosome-mediated processing of Gli3, and the ratio of Gli3 repressor to full-length (activator) form of Gli3 determines the Gli3 repressor output required for normal eye development. Our results suggest Zic5 is a critical player in regulating Gli3 stability for the proper differentiation of RPE and rod photoreceptor layer during Xenopus eye development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022. Published by Elsevier Inc.)

Pattern formation of biological structures involves the arrangement of different types of cells in an ordered spatial configuration. In this study, we investigate the mechanism of patterning the Drosophila eye epithelium into a precise triangular grid of photoreceptor clusters called ommatidia. Previous studies had led to a long-standing biochemical model whereby a reaction-diffusion process is templated by recently formed ommatidia to propagate a molecular prepattern across the eye. Here, we find that the templating mechanism is instead, mechanochemical in origin; newly born columns of differentiating ommatidia serve as a template to spatially pattern flows that move epithelial cells into position to form each new column of ommatidia. Cell flow is generated by a source and sink, corresponding to narrow zones of cell dilation and contraction respectively, that straddle the growing wavefront of ommatidia. The newly formed lattice grid of ommatidia cells are immobile, deflecting, and focusing the flow of other cells. Thus, the self-organization of a regular pattern of cell fates in an epithelium is mechanically driven., Competing Interests: KG, MM, RC No competing interests declared, (© 2022, Gallagher et al.)

Angiogenesis is a stepwise process leading to blood vessel formation. In the vertebrate retina, endothelial cells are guided by astrocytes migrating along the inner surface, and the two processes are coupled by a tightly regulated cross-talks between the two cell types. Here, I have investigated how the FAT1 cadherin, a regulator of tissue morphogenesis that governs tissue cross-talk, influences retinal vascular development. Late-onset Fat1 inactivation in the neural lineage in mice, by interfering with astrocyte progenitor migration polarity and maturation, delayed postnatal retinal angiogenesis, leading to persistent vascular abnormalities in adult retinas. Impaired astrocyte migration and polarity were not associated with alterations of retinal ganglion cell axonal trajectories or of the inner limiting membrane. In contrast, inducible Fat1 ablation in postnatal astrocytes was sufficient to alter their migration polarity and proliferation. Altogether, this study uncovers astrocyte-intrinsic and -extrinsic Fat1 activities that influence astrocyte migration polarity, proliferation and maturation, disruption of which impacts retinal vascular development and maintenance., Competing Interests: Competing interests The author declares no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Neural activity has been implicated in the motility and outgrowth of glial cell processes throughout the central nervous system. Here, we explore this phenomenon in Müller glia, which are specialized radial astroglia that are the predominant glial type of the vertebrate retina. Müller glia extend fine filopodia-like processes into retinal synaptic layers, in similar fashion to brain astrocytes and radial glia that exhibit perisynaptic processes. Using two-photon volumetric imaging, we found that during the second postnatal week, Müller glial processes were highly dynamic, with rapid extensions and retractions that were mediated by cytoskeletal rearrangements. During this same stage of development, retinal waves led to increases in cytosolic calcium within Müller glial lateral processes and stalks. These regions comprised distinct calcium compartments, distinguished by variable participation in waves, timing, and sensitivity to an M1 muscarinic acetylcholine receptor antagonist. However, we found that motility of lateral processes was unaffected by the presence of pharmacological agents that enhanced or blocked wave-associated calcium transients. Finally, we found that mice lacking normal cholinergic waves in the first postnatal week also exhibited normal Müller glial process morphology. Hence, outgrowth of Müller glial lateral processes into synaptic layers is determined by factors that are independent of neuronal activity., Competing Interests: JT, CC No competing interests declared, MF Reviewing editor, eLife, (© 2021, Tworig et al.)

Microglia are brain-resident macrophages that function as the first line of defense in brain. Embryonic microglial precursors originate in peripheral mesoderm and migrate into the brain during development. However, the mechanism by which they colonize the brain is incompletely understood. The retina is one of the first brain regions to accommodate microglia. In zebrafish, embryonic microglial precursors use intraocular hyaloid blood vessels as a pathway to migrate into the optic cup via the choroid fissure. Once retinal progenitor cells exit the cell cycle, microglial precursors associated with hyaloid blood vessels start to infiltrate the retina preferentially through neurogenic regions, suggesting that colonization of retinal tissue depends upon the neurogenic state. Along with blood vessels and retinal neurogenesis, IL34 also participates in microglial precursor colonization of the retina. Altogether, CSF receptor signaling, blood vessels, and neuronal differentiation function as cues to create an essential path for microglial migration into developing retina., Competing Interests: NR, IM No competing interests declared, (© 2021, Ranawat and Masai.)

Microglia are resident immune cells in the central nervous system, showing a regular distribution. Advancing microscopy and image processing techniques have contributed to elucidating microglia's morphology, dynamics, and distribution. However, the mechanism underlying the regular distribution of microglia remains to be elucidated. First, we quantitatively confirmed the regularity of the distribution pattern of microglial soma in the retina. Second, we formulated a mathematical model that includes factors that may influence regular distribution. Next, we experimentally quantified the model parameters (cell movement, process formation, and ATP dynamics). The resulting model simulation from the measured parameters showed that direct cell-cell contact is most important in generating regular cell spacing. Finally, we tried to specify the molecular pathway responsible for the repulsion between neighboring microglia., (© 2021. The Author(s).)

Actomyosin contraction shapes the Drosophila eye's panoramic view. The convex curvature of the retinal epithelium, organized in ∼800 close-packed ommatidia, depends upon a fourfold condensation of the retinal floor mediated by contraction of actin stress fibers in the endfeet of interommatidial cells (IOCs). How these tensile forces are coordinated is not known. Here, we discover a previously unobserved phenomenon: Ca2+ waves regularly propagate across the IOC network in pupal and adult eyes. Genetic evidence demonstrates that IOC waves are independent of phototransduction, but require the inositol 1,4,5-triphosphate receptor (IP3R), suggesting that these waves are mediated by Ca2+ releases from endoplasmic reticulum stores. Removal of IP3R disrupts stress fibers in IOC endfeet and increases the basal retinal surface by ∼40%, linking IOC waves to facilitation of stress fiber contraction and floor morphogenesis. Furthermore, IP3R loss disrupts the organization of a collagen IV network underneath the IOC endfeet, implicating the extracellular matrix and its interaction with stress fibers in eye morphogenesis. We propose that coordinated cytosolic Ca2+ increases in IOC waves promote stress fiber contractions, ensuring an organized application of the planar tensile forces that condense the retinal floor. This article has an associated 'The people behind the papers' interview., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Rhodopsin ( RHO ) misfolding mutations are a common cause of the blinding disease autosomal dominant retinitis pigmentosa (adRP). The most prevalent mutation, RHO P23H , results in its misfolding and retention in the endoplasmic reticulum (ER). Under homeostatic conditions, misfolded proteins are selectively identified, retained at the ER, and cleared via ER-associated degradation (ERAD). Overload of these degradation processes for a prolonged period leads to imbalanced proteostasis and may eventually result in cell death. ERAD of misfolded proteins, such as RHO P23H , includes the subsequent steps of protein recognition, targeting for ERAD, retrotranslocation, and proteasomal degradation. In the present study, we investigated and compared pharmacological modulation of ERAD at these four different major steps. We show that inhibition of the VCP/proteasome activity favors cell survival and suppresses P23H-mediated retinal degeneration in RHO P23H rat retinal explants. We suggest targeting this activity as a therapeutic approach for patients with currently untreatable adRP.

Extracellular vesicles (EVs), which consist of microvesicles and exosomes, are secreted from all cells to transform vital information in the form of lipids, proteins, mRNAs and small RNAs such as microRNAs (miRNAs). Many studies demonstrated that EVs' miRNAs have effects on target cells. Numerous people suffer from the blindness caused by retinal degenerations. The death of retinal neurons is irreversible and creates permanent damage to the retina. In the absence of acceptable cures for retinal degenerative diseases, stem cells and their paracrine agents including EVs have become a promising therapeutic approach. Several studies showed that the therapeutic effects of stem cells are due to the miRNAs of their EVs. Considering the effects of microRNAs in retinal cells development and function and studies which provide the possible roles of mesenchymal stem cells-derived EVs miRNA content on retinal diseases, we focused on the similarities between these two groups of miRNAs that could be helpful for promoting new therapeutic techniques for retinal degenerative diseases., (© 2021. The Author(s).)

Purpose: The purpose of this study was to develop an in vivo optical coherence tomography (OCT) system capable of imaging the developing mouse retina and its associated morphometric and microstructural changes., Methods: Thirty-four wild-type mice (129S1/SvlmJ) were anesthetized and imaged between postnatal (P) day 7 and P21. OCT instrumentation was developed to optimize signal intensity and image quality. Semi-automatic segmentation tools were developed to quantify the retinal thickness of the nerve fiber layer (NFL), inner plexiform layer (IPL), inner nuclear layer (INL), and the outer retinal layers (ORL), in addition to the total retina. The retinal maturation was characterized by comparing layer thicknesses between consecutive time points., Results: From P7 to P10, the IPL increased significantly, consistent with retinal synaptogenesis. From P10 to P12, the IPL and ORL also increased, which is coherent with synaptic connectivity and photoreceptor maturation. In contrast, during these periods, the INL decreased significantly, consistent with cellular densification and selective apoptotic "pruning" of the tissue during nuclear migration. Thereafter from P12 to P21, the INL continued to thin (significantly from P17 to P21) whereas the other layers remained unchanged. No time-dependent changes were observed in the NFL. Overall, changes in the total retina were attributed to those in the IPL, INL, and ORL. Regions of the retina adjacent to the optic nerve head were thinner than distal regions during maturation., Conclusions: Changes in retinal layer thickness are consistent with retinal developmental mechanisms. Accordingly, this report opens new horizons in using our system in the mouse to characterize longitudinally developmental digressions in models of human diseases.

Human retina development involves multiple well-studied signaling pathways that promote the genesis of a wide arrange of different cell types in a complex architectural structure. Human embryonic stem cells (hESCs)-derived retinal organoids could recapitulate the human retinal development. We performed single-cell RNA-seq of retinal organoids from 5 time points (D36, D66, D96, D126, D186) and identified 9 distinct populations of cells. In addition, we analyzed the molecular characteristics of each main population and followed them from genesis to maturity by pseudotime analysis and characterized the cell-cell interactions between different cell types. Interestingly, we identified insulin receptor (INSR) as a specifically expressed receptor involved in the genesis of photoreceptors, and pleiothropin (PTN)-protein tyrosine phosphatase receptor type Z1 (PTPRZ1) as a mediator of a previously unknown interaction between Müller and retinal progenitor cells. Taken together, these findings provide a rich transcriptome-based lineage map for studying human retinal development and modeling developmental disorders in retinal organoids., (© 2021. Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature.)

DNA methylation is important in developing and post-mitotic cells in various tissues. Recent studies have shown that DNA methylation is highly dynamic, and plays important roles during retinal development and aging. In addition, the dynamic regulation of DNA methylation is involved in the occurrence and development of age-related macular degeneration and diabetic retinopathy and shows potential in disease diagnoses and prognoses. This review introduces the epigenetic concepts of DNA methylation and demethylation with an emphasis on their regulatory roles in retinal development and related diseases. Moreover, we propose exciting ideas such as its crosstalk with other epigenetic modifications and retinal regeneration, to provide a potential direction for understanding retinal diseases from the epigenetic perspective., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Recent advances in high throughput single-cell RNA sequencing (scRNA-seq) technology have enabled the simultaneous transcriptomic profiling of thousands of individual cells in a single experiment. To investigate the intrinsic process of retinal development, researchers have leveraged this technology to quantify gene expression in retinal cells across development, in multiple species, and from numerous important models of human disease. In this review, we summarize recent applications of scRNA-seq and discuss how these datasets have complemented and advanced our understanding of retinal progenitor cell competence, cell fate specification, and differentiation. Finally, we also highlight the outstanding questions in the field that advances in single-cell data generation and analysis will soon be able to answer., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Throughout the central nervous system, astrocytes adopt precisely ordered spatial arrangements of their somata and arbors, which facilitate their many important functions. Astrocyte pattern formation is particularly important in the retina, where astrocytes serve as a template that dictates the pattern of developing retinal vasculature. Thus, if astrocyte patterning is disturbed, there are severe consequences for retinal angiogenesis and ultimately for vision - as seen in diseases such as retinopathy of prematurity. Here we discuss key steps in development of the retinal astrocyte population. We describe how fundamental developmental forces - their birth, migration, proliferation, and death - sculpt astrocytes into a template that guides angiogenesis. We further address the radical changes in the cellular and molecular composition of the astrocyte network that occur upon completion of angiogenesis, paving the way for their adult functions in support of retinal ganglion cell axons. Understanding development of retinal astrocytes may elucidate pattern formation mechanisms that are deployed broadly by other axon-associated astrocyte populations., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Human organoids stand at the forefront of basic and translational research, providing experimentally tractable systems to study human development and disease. These stem cell-derived, in vitro cultures can generate a multitude of tissue and organ types, including distinct brain regions and sensory systems. Neural organoid systems have provided fundamental insights into molecular mechanisms governing cell fate specification and neural circuit assembly and serve as promising tools for drug discovery and understanding disease pathogenesis. In this review, we discuss several human neural organoid systems, how they are generated, advances in 3D imaging and bioengineering, and the impact of organoid studies on our understanding of the human nervous system., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

In 1994, Burrill and Easter described the retinal projections in embryonic and larval zebrafish, introducing the term "arborization fields" (AFs) for the retinorecipient areas. AFs were numbered from 1 to 10 according to their positions along the optic tract. With the exception of AF10 (neuropil of the optic tectum), annotations of AFs remained tentative. Here we offer an update on the likely identities and functions of zebrafish AFs after successfully matching classical neuroanatomy to the digital Max Planck Zebrafish Brain Atlas. In our system, individual AFs are neuropil areas associated with the following nuclei: AF1 with the suprachiasmatic nucleus; AF2 with the posterior parvocellular preoptic nucleus; AF3 and AF4 with the ventrolateral thalamic nucleus; AF4 with the anterior and intermediate thalamic nuclei; AF5 with the dorsal accessory optic nucleus; AF7 with the parvocellular superficial pretectal nucleus; AF8 with the central pretectal nucleus; and AF9d and AF9v with the dorsal and ventral periventricular pretectal nuclei. AF6 is probably part of the accessory optic system. Imaging, ablation, and activation experiments showed contributions of AF5 and potentially AF6 to optokinetic and optomotor reflexes, AF4 to phototaxis, and AF7 to prey detection. AF6, AF8 and AF9v respond to dimming, and AF4 and AF9d to brightening. While few annotations remain tentative, it is apparent that the larval zebrafish visual system is anatomically and functionally continuous with its adult successor and fits the general cyprinid pattern. This study illustrates the synergy created by merging classical neuroanatomy with a cellular-resolution digital brain atlas resource and functional imaging in larval zebrafish., (© 2021 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals LLC.)

The growth of the mouse eye and retina after birth is a dynamic, highly regulated process. In this study, we applied visible-light optical coherence tomography (vis-OCT), a non-invasive imaging technique, to examine developing retinal layer structures after eye-opening. We introduced a resampled circumpapillary B-scan averaging technique to improve the inter-layer contrast, enabling retinal layer thickness measurements as early as postnatal day 13 (P13) - right after eye-opening. We confirmed vis-OCT measurements using ex vivo confocal microscopy of retinal sections at different ages. Our results demonstrate that vis-OCT can visualize the developmental murine retinal layer structure in vivo, which offers us new opportunities to better characterize the pathological alterations in mouse models of developmental eye diseases., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Retinal development is tightly regulated to ensure the generation of appropriate cell types and the assembly of functional neuronal circuitry. Despite remarkable advances have been made in understanding regulation of gene expression during retinal development, how translational regulation guides retinogenesis is less understood. Here, we conduct a comprehensive translatome and transcriptome survey to the mouse retinogenesis from the embryonic to the adult stages. We discover thousands of genes that have dynamic changes at the translational level and pervasive translational regulation in a developmental stage-specific manner with specific biological functions. We further identify genes whose translational efficiencies are frequently controlled by changing usage in upstream open reading frame during retinal development. These genes are enriched for biological functions highly important to neurons, such as neuron projection organization and microtubule-based protein transport. Surprisingly, we discover hundreds of previously uncharacterized micropeptides, translated from putative long non-coding RNAs and circular RNAs. We validate their protein products in vitro and in vivo and demonstrate their potentials in regulating retinal development. Together, our study presents a rich and complex landscape of translational regulation and provides novel insights into their roles during retinogenesis., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Modern western diets have been associated with a reduced proportion of dietary omega-3 fatty acids leading to decreased levels of DHA (docosahexaenoic acid) in the brain. Low DHA content has been associated with altered development of visual acuity in infants and also with an altered time course of synapse elimination and plasticity in subcortical visual nuclei in rodents. Microglia has an active role in normal developmental processes such as circuitry refinement and plasticity, and its activation status can be modulated by omega-3 (ω3) and omega-6 (ω6) essential fatty acids. In the present study, we investigated the impact of dietary restriction of DHA (ω3 - ), through the chronic administration of a coconut-based diet as the only fat source. This dietary protocol resulted in a reduction in DHA content in the retina and superior colliculus (SC) and in a neuroinflammatory outcome during the development of the rodent visual system. The ω3 - group showed changes in microglial morphology in the retina and SC and a corresponding altered pattern of pro-inflammatory cytokine expression. Early and late fish oil protocols supplementation were able to restore DHA levels. The early supplementation also decreased neuroinflammatory markers in the visual system. The present study indicates that a chronic dietary restriction of omega-3 fatty acids and the resulting deficits in DHA content, commonly observed in Western diets, interferes with the microglial profile leading to an inflamed microenvironment which may underlie a disruption of synapse elimination, altered topographical organization, abnormal plasticity, and duration of critical periods during brain development., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Myopia (short-sightedness), usually caused by excessive elongation of the eye during development, has reached epidemic proportions worldwide. In animal systems including the chicken model, several treatments have been shown to inhibit ocular elongation and experimental myopia. Although diverse in their apparent mechanism of action, each one leads to a reduction in the rate of ocular growth. We hypothesize that a defined set of retinal molecular changes may underlie growth inhibition, irrespective of the treatment agent used. Accordingly, across five well-established but diverse methods of inhibiting myopia, significant overlap is seen in the retinal transcriptome profile (transcript levels and alternative splicing events) in chicks when analyzed by RNA-seq. Within the two major pathway networks enriched during growth inhibition, that of cell signaling and circadian entrainment, transcription factors form the largest functional grouping. Importantly, a large percentage of those genes forming the defined retinal response are downstream targets of the transcription factor EGR1 which itself shows a universal response to all five growth-inhibitory treatments. This supports EGR1's previously implicated role in ocular growth regulation. Finally, by contrasting our data with human linkage and GWAS studies on refractive error, we confirm the applicability of our study to the human condition. Together, these findings suggest that a universal set of transcriptome changes, which sit within a well-defined retinal network that cannot be bypassed, is fundamental to growth regulation, thus paving a way for designing novel targets for myopia therapies., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

The visual system is the best system to study activity-dependent sensory circuit development. The connections from the retina to the dorsal lateral geniculate nucleus, the retinogeniculate connections, undergo extensive remodeling during early postnatal life. Thus, techniques that allow the expression of transgenes early in the developing retina are essential to study visual system development. Here, we describe a protocol to express genes-of-interest in the developing mouse retina via in utero intraocular adeno-associated virus injections. For complete details on the use and execution of this protocol, please refer to Yasuda et al. (2021)., Competing Interests: The authors declare no competing interest., (© 2021 The Author(s).)

Photoreceptors are critical components of the retina and play a role in the first step of the conversion of light to electric signals. With the discovery of the intrinsically photosensitive retinal ganglion cells, which regulate non-image-forming visual processes, our knowledge of the photosensitive cell family in the retina has deepened. Photoreceptor development is regulated by specific genes and proteins and involves a series of molecular processes including DNA transcription, post-transcriptional modification, protein translation, and post-translational modification. Single-cell sequencing is a promising technology for the study of photoreceptor development. This review presents an overview of the types of human photoreceptors, summarizes recent discoveries in the regulatory mechanisms underlying their development at single-cell resolution, and outlines the prospects in this field.

Background: In retinal degenerative disease, progressive and debilitating conditions result in deterioration of retinal cells and visual loss. In human, retina lacks the inherent capacity for regeneration. Therefore, regeneration of retinal layer from human retinal progenitor cells (hRPCs) is a challenging task and restricted in vitro maintenance of hRPCs remains as the main hurdle. Retina and anterior neural fold homeobox gene (RAX) play critical roles in developing retina and maintenance of hRPCs. In this study, for the first time regulatory regions of human RAX gene with potential promoter activity were experimentally investigated., Results: For this purpose, after in silico analysis of regulatory regions of human RAX gene, the expression of EGFP reporter derived by putative promoter sequences was first evaluated in 293 T cells and then in hRPCS derived from human embryonic stem cells. The candidate region (RAX-3258 bp) showed the highest EGFP expression in hRPCs. This reporter construct can be used for in vitro monitoring of hRPC identity and verification of an efficient culture medium for maintenance of these cells., Conclusions: Furthermore, our findings provide a platform for better insight into regulatory regions of human RAX gene and molecular mechanisms underlying its vital functions in retina development., (© 2021. The Author(s).)

The adult lamprey retina has two types of photoreceptor cells, short and long photoreceptor cells, which are equivalent to rods and cones of other vertebrates. In contrast, the retina of lamprey larvae only contains a single type of photoreceptor cell, which appears to correspond to the short photoreceptor cell. However, the developmental pattern of the long photoreceptor cell is unknown. Previously, we reported that antibodies against rhodopsin and iodopsin (the chicken red cone opsin) could discriminate between the outer segments of short and long photoreceptor cells, respectively, in the retina of adult Japanese river lamprey ( Lethenteron camtschaticum ). Here, we immunohistochemically investigate the appearance of long photoreceptor cells in the larval and adult retinas of the Far Eastern brook lamprey ( Lethenteron reissneri ), which is a close relative of the Japanese river lamprey, by using anti-iodopsin antibody. We found that iodopsin immunoreactivity was localized not only in the adult retina but also in the larval retina. Moreover, we examined the immunohistochemical localization of signal transduction molecules, such as transducin and arrestin, in the iodopsin-immunoreactive cells of the larval retina. The iodopsin-immunoreactive cells also contained both transducin and arrestin, suggesting that long photoreceptor cells are already functional in the larval stage before the acquisition of visual function. Our results suggest that the iodopsin-immunoreactive cells may be related to not only cone vision in the adult but also photoreception in the larval lamprey.

Visual opsins are proteins expressed by retinal photoreceptors that capture light to begin the process of phototransduction. In vertebrates, the two types of photoreceptors (rods and cones) express one or multiple opsins and are distributed in variable patterns across the retina. Some cones form opsin retinal gradients, as in the mouse, whereas others form more demarcated opsin domains, as in the lattice-like mosaic retinas of teleost fishes. Reduced rod opsin (rh1) expression in mouse, zebrafish, and African clawed frog results in lack of photoreceptor outer segments (i.e., the cilium that houses the opsins) and, in the case of the mouse, to retinal degeneration. The effects of diminished cone opsin expression have only been studied in the mouse where knockout of the short-wavelength sensitive 1 (sws1) opsin leads to ventral retinal cones lacking outer segments, but no retinal degeneration. Here we show that, following CRISPR/Cas9 injections that targeted knockout of the sws1 opsin in rainbow trout, fish with diminished sws1 opsin expression exhibited a variety of developmental defects including head and eye malformations, underdeveloped outer retina, mislocalized opsin expression, cone degeneration, and mosaic irregularity. All photoreceptor types were affected even though sws1 is only expressed in the single cones of wild fish. Our results reveal unprecedented developmental defects associated with diminished cone opsin expression and suggest that visual opsin genes are involved in regulatory processes that precede photoreceptor differentiation., (© 2021 Wiley Periodicals LLC.)

The ability to perceive and respond to environmental stimuli emerges in the absence of sensory experience. Spontaneous retinal activity prior to eye opening guides the refinement of retinotopy and eye-specific segregation in mammals, but its role in the development of higher-order visual response properties remains unclear. Here, we describe a transient window in neonatal mouse development during which the spatial propagation of spontaneous retinal waves resembles the optic flow pattern generated by forward self-motion. We show that wave directionality requires the same circuit components that form the adult direction-selective retinal circuit and that chronic disruption of wave directionality alters the development of direction-selective responses of superior colliculus neurons. These data demonstrate how the developing visual system patterns spontaneous activity to simulate ethologically relevant features of the external world and thereby instruct self-organization., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Organoids derived from pluripotent stem cells promise the solution to current challenges in basic and biomedical research. Mammalian organoids are however limited by long developmental time, variable success, and lack of direct comparison to an in vivo reference. To overcome these limitations and address species-specific cellular organization, we derived organoids from rapidly developing teleosts. We demonstrate how primary embryonic pluripotent cells from medaka and zebrafish efficiently assemble into anterior neural structures, particularly retina. Within 4 days, blastula-stage cell aggregates reproducibly execute key steps of eye development: retinal specification, morphogenesis, and differentiation. The number of aggregated cells and genetic factors crucially impacted upon the concomitant morphological changes that were intriguingly reflecting the in vivo situation. High efficiency and rapid development of fish-derived organoids in combination with advanced genome editing techniques immediately allow addressing aspects of development and disease, and systematic probing of impact of the physical environment on morphogenesis and differentiation., Competing Interests: LZ, VW, TT, CS, TT, JW No competing interests declared, (© 2021, Zilova et al.)