The Analysis of Embryoid Body Formation and Its Role in Retinal Organoid Development.

Within the last decade, a wide variety of protocols have emerged for the generation of retinal organoids. A subset of studies have compared protocols based on stem cell source, the physical features of the microenvironment, and both internal and external signals, all features that influence embryoid body and retinal organoid formation. Most of these comparisons have focused on the effect of signaling pathways on retinal organoid development. In this study, our aim is to understand whether starting cell conditions, specifically those involved in embryoid body formation, affect the development of retinal organoids in terms of differentiation capacity and reproducibility. To investigate this, we used the popular 3D floating culture method to generate retinal organoids from stem cells. This method starts with either small clumps of stem cells generated from larger clones (clumps protocol, CP) or with an aggregation of single cells (single cells protocol, SCP). Using histological analysis and gene-expression comparison, we found a retention of the pluripotency capacity on embryoid bodies generated through the SCP compared to the CP. Nonetheless, these early developmental differences seem not to impact the final retinal organoid formation, suggesting a potential compensatory mechanism during the neurosphere stage. This study not only facilitates an in-depth exploration of embryoid body development but also provides valuable insights for the selection of the most suitable protocol in order to study retinal development and to model inherited retinal disorders in vitro.

Retinal Development in a Precocial Bird Species, the Quail (Coturnix coturnix, Linnaeus 1758).

The quail (Coturnix coturnix, Linnaeus 1758), a notable model used in developmental biology, is a precocial bird species in which the processes of retinal cell differentiation and retinal histogenesis have been poorly studied. The purpose of the present research is to examine the retinogenesis in this bird species immunohistochemically and compare the results with those from previous studies in precocial and altricial birds. We found that the first PCNA-negative nuclei are detected at Stage (St) 21 in the vitreal region of the neuroblastic layer, coinciding topographically with the first αTubAc-/Tuj1-/Isl1-immunoreactive differentiating ganglion cells. At St28, the first Prox1-immunoreactive nuclei can be distinguished in the vitreal side of the neuroblastic layer (NbL), but also the first visinin-immunoreactive photoreceptors in the scleral surface. The inner plexiform layer (IPL) emerges at St32, and the outer plexiform layer (OPL) becomes visible at St35-the stage in which the first GS-immunoreactive Müller cells are distinguishable. Newly hatched animals show a well-developed stratified retina in which the PCNA-and pHisH3-immunoreactivies are absent. Therefore, retinal cell differentiation in the quail progresses in the stereotyped order conserved among vertebrates, in which ganglion cells initially appear and are followed by amacrine cells, horizontal cells, and photoreceptors. Müller glia are one of the last cell types to be born. Plexiform layers emerge following a vitreal-to-scleral gradient. Finally, our results suggest that there are no significant differences in the timing of different events involved in retinal maturation between the quail and the chicken, but the same events are delayed in an altricial bird species.

Retinal neuroblast migration and ganglion cell layer organization require the cytoskeletal-interacting protein Mllt11.

BACKGROUND: The vertebrate retina is an organized laminar structure comprised of distinct cell types populating three nuclear layers. During development, each retinal cell type follows a stereotypical temporal order of genesis, differentiation, and migration, giving rise to its stratified organization. Once born, the precise positioning of cells along the apico-basal (radial) axis of the retina is critical for subsequent connections to form, relying on highly orchestrated migratory processes. While these processes are critical for visual function to arise, the regulators of cellular migration and retinal lamination remain largely unexplored. RESULTS: We report a role for a microtubule-interacting protein, Mllt11 (myeloid/lymphoid or mixed-lineage leukemia; translocated to chromosome 11/All1 fused gene from chromosome 1q) in mammalian retinal cell migration during retinogenesis. We show that Mllt11 loss-of-function in mouse retinal neuroblasts affected the migration of ganglion and amacrine cells into the ganglion cell layer and led to their aberrant accumulation in the inner nuclear and plexiform layers. CONCLUSIONS: We demonstrate a role for Mllt11 in neuroblast migration and formation of the ganglion cell layer of the retina.

Starvation to Glucose Reprograms Development of Neurovascular Unit in Embryonic Retinal Cells.

Perinatal exposure to starvation is a risk factor for development of severe retinopathy in adult patients with diabetes. However, the underlying mechanisms are not completely understood. In the present study, we shed light on molecular consequences of exposure to short-time glucose starvation on the transcriptome profile of mouse embryonic retinal cells. We found a profound downregulation of genes regulating development of retinal neurons, which was accompanied by reduced expression of genes encoding for glycolytic enzymes and glutamatergic signaling. At the same time, glial and vascular markers were upregulated, mimicking the diabetes-associated increase of angiogenesis-a hallmark of pathogenic features in diabetic retinopathy. Energy deprivation as a consequence of starvation to glucose seems to be compensated by upregulation of genes involved in fatty acid elongation. Results from the present study demonstrate that short-term glucose deprivation during early fetal life differentially alters expression of metabolism- and function-related genes and could have detrimental and lasting effects on gene expression in the retinal neurons, glial cells, and vascular elements and thus potentially disrupting gene regulatory networks essential for the formation of the retinal neurovascular unit. Abnormal developmental programming during retinogenesis may serve as a trigger of reactive gliosis, accelerated neurodegeneration, and increased vascularization, which may promote development of severe retinopathy in patients with diabetes later in life.

Activation of adult mammalian retinal stem cells in vivo via antagonism of BMP and sFRP2.

BACKGROUND: The adult mammalian retina does not have the capacity to regenerate cells lost due to damage or disease. Therefore, retinal injuries and blinding diseases result in irreversible vision loss. However, retinal stem cells (RSCs), which participate in retinogenesis during development, persist in a quiescent state in the ciliary epithelium (CE) of the adult mammalian eye. Moreover, RSCs retain the ability to generate all retinal cell types when cultured in vitro, including photoreceptors. Therefore, it may be possible to activate endogenous RSCs to induce retinal neurogenesis in vivo and restore vision in the adult mammalian eye. METHODS: To investigate if endogenous RSCs can be activated, we performed combinatorial intravitreal injections of antagonists to BMP and sFRP2 proteins (two proposed mediators of RSC quiescence in vivo), with or without growth factors FGF and Insulin. We also investigated the effects of chemically-induced N-methyl-N-Nitrosourea (MNU) retinal degeneration on RSC activation, both alone and in combination withthe injected factors. Further, we employed inducible Msx1-CreERT2 genetic lineage labeling of the CE followed by stimulation paradigms to determine if activated endogenous RSCs could migrate into the retina and differentiate into retinal neurons. RESULTS: We found that in vivo antagonism of BMP and sFRP2 proteins induced CE cells in the RSC niche to proliferate and expanded the RSC population. BMP and sFRP2 antagonism also enhanced CE cell proliferation in response to exogenous growth factor stimulation and MNU-induced retinal degeneration. Furthermore, Msx1-CreERT2 genetic lineage tracing revealed that CE cells migrated into the retina following stimulation and/or injury, where they expressed markers of mature photoreceptors and retinal ganglion cells. CONCLUSIONS: Together, these results indicate that endogenous adult mammalian RSCs may have latent regenerative potential that can be activated by modulating the RSC niche and hold promise as a means for endogenous retinal cell therapy to repair the retina and improve vision.

Generation and Staging of Human Retinal Organoids Based on Self-Formed Ectodermal Autonomous Multi-Zone System.

Methods for stem cell-derived, three-dimensional retinal organoids induction have been established and shown great potential for retinal development modeling and drug screening. Herein, we reported an exogenous-factors-free and robust method to generate retinal organoids based on "self-formed ectodermal autonomous multi-zone" (SEAM) system, a two-dimensional induction scheme that can synchronously generate multiple ocular cell lineages. Characterized by distinct morphological changes, the differentiation of the obtained retinal organoids could be staged into the early and late differentiation phases. During the early differentiation stage, retinal ganglion cells, cone photoreceptor cells (PRs), amacrine cells, and horizontal cells developed; whereas rod PRs, bipolar cells, and Müller glial cells were generated in the late differentiation phase, resembling early-phase and late-phase retinogenesis in vivo. Additionally, we modified the maintenance strategy for the retinal organoids and successfully promoted their long-term survival. Using 3D immunofluorescence image reconstruction and transmission electron microscopy, the substantial mature PRs with outer segment, inner segment and ribbon synapse were demonstrated. Besides, the retinal pigment epithelium (RPE) was induced with distinct boundary and the formation of ciliary margin was observed by co-suspending retina organoids with the zone containing RPE. The obtained RPE could be expanded and displayed similar marker expression, ultrastructural feature and functional phagocytosis to native RPE. Thus, this research described a simple and robust system which enabled generation of retina organoids with substantial mature PRs, RPE and the ciliary margin without the need of exogenous factors, providing a new platform for research of retinogenesis and retinal translational application.

Quantitative Approaches to Study Retinal Neurogenesis.

The study of the development of the vertebrate retina can be addressed from several perspectives: from a purely qualitative to a more quantitative approach that takes into account its spatio-temporal features, its three-dimensional structure and also the regulation and properties at the systems level. Here, we review the ongoing transition toward a full four-dimensional characterization of the developing vertebrate retina, focusing on the challenges at the experimental, image acquisition, image processing and quantification. Using the developing zebrafish retina, we illustrate how quantitative data extracted from these type of highly dense, three-dimensional tissues depend strongly on the image quality, image processing and algorithms used to segment and quantify. Therefore, we propose that the scientific community that focuses on developmental systems could strongly benefit from a more detailed disclosure of the tools and pipelines used to process and analyze images from biological samples.

Adherent but Not Suspension-Cultured Embryoid Bodies Develop into Laminated Retinal Organoids.

Human induced pluripotent stem cells (iPSCs) are differentiated into three-dimensional (3D) retinal organoids to study retinogenesis and diseases that would otherwise be impossible. The complexity and low yield in current protocols remain a technical challenge, particularly for inexperienced personnel. Differentiation protocols require labor-intensive and time-consuming dissection of optic vesicles (OVs). Here we compare this method with a suspension method of developing retinal organoids. iPSCs were differentiated with standard protocols but the suspension-grown method omitted the re-plating of embryoid bodies and dissection of OVs. All other media and treatments were identical between developmental methods. Developmental maturation was evaluated with RT-qPCR and immunocytochemistry. Dissection- and suspension-derived retinal organoids displayed temporal biogenesis of retinal cell types. Differences in retinal organoids generated by the two methods of differentiation included temporal developmental and the organization of neural retina layers. Retinal organoids grown in suspension showed delayed development and disorganized retinal layers compared to the dissected retinal organoids. We found that omitting the re-plating of EBs to form OVs resulted in numerous OVs that were easy to identify and matured along a retinal lineage. While more efficient, the suspension method led to retinal organoids with disorganized retinal layers compared to those obtained using conventional dissection protocols.

The Role of Small Molecules and Their Effect on the Molecular Mechanisms of Early Retinal Organoid Development.

Early in vivo embryonic retinal development is a well-documented and evolutionary conserved process. The specification towards eye development is temporally controlled by consecutive activation or inhibition of multiple key signaling pathways, such as the Wnt and hedgehog signaling pathways. Recently, with the use of retinal organoids, researchers aim to manipulate these pathways to achieve better human representative models for retinal development and disease. To achieve this, a plethora of different small molecules and signaling factors have been used at various time points and concentrations in retinal organoid differentiations, with varying success. Additions differ from protocol to protocol, but their usefulness or efficiency has not yet been systematically reviewed. Interestingly, many of these small molecules affect the same and/or multiple pathways, leading to reduced reproducibility and high variability between studies. In this review, we make an inventory of the key signaling pathways involved in early retinogenesis and their effect on the development of the early retina in vitro. Further, we provide a comprehensive overview of the small molecules and signaling factors that are added to retinal organoid differentiation protocols, documenting the molecular and functional effects of these additions. Lastly, we comparatively evaluate several of these factors using our established retinal organoid methodology.

Distribution of planar cell polarity proteins in the developing avian retina.

Planar cell polarity (PCP) is evolutionary conserved and play a critical role in proper tissue development and function. During central nervous system development, PCP proteins exhibit specific patterns of distribution and are indispensable for axonal growth, dendritogenesis, neuronal migration, and neuronal differentiation. The retina constitutes an excellent model in which to study molecular mechanisms involved in neural development. The analysis of the spatiotemporal expression of PCP proteins in this model constitutes an useful histological approach in order to identify possible roles of these proteins in retinogenesis. Immunohistochemical techniques revealed that Frz6, Celsr1, Vangl1, Pk1, Pk3, and Fat1 were present in emerging axons from recently differentiated ganglion cells in the chicken retina. Except for Vangl1, they were also asymmetrically distributed in differentiated amacrine cells. Pk1 and Pk3 were restricted in the outer nuclear layer to the outer segment of photoreceptors. Vangl1 was also located in the cell somata of Müller glia. Given these findings together, the distribution of PCP proteins in the developing chicken retina suggest essential roles in axonal guidance during early retinogenesis and a possible involvement in the establishment of cell asymmetry and maintenance of retinal cell phenotypes.

Analysis of Programmed Cell Death and Senescence Markers in the Developing Retina of an Altricial Bird Species.

This study shows the distribution patterns of apoptotic cells and biomarkers of cellular senescence during the ontogeny of the retina in the zebra finch (T. guttata). Neurogenesis in this altricial bird species is intense in the retina at perinatal and post-hatching stages, as opposed to precocial bird species in which retinogenesis occurs entirely during the embryonic period. Various phases of programmed cell death (PCD) were distinguishable in the T. guttata visual system. These included areas of PCD in the central region of the neuroretina at the stages of optic cup morphogenesis, and in the sub-optic necrotic centers (St15-20). A small focus of early neural PCD was detected in the neuroblastic layer, dorsal to the optic nerve head, coinciding with the appearance of the first differentiated neuroblasts (St24-St25). There were sparse pyknotic bodies in the non-laminated retina between St26 and St37. An intense wave of neurotrophic PCD was detected in the laminated retina between St42 and P8, the last post-hatching stage included in the present study. PCD was absent from the photoreceptor layer. Phagocytic activity was also detected in Müller cells during the wave of neurotrophic PCD. With regard to the chronotopographical staining patterns of senescence biomarkers, there was strong parallelism between the SA-ß-GAL signal and p21 immunoreactivity in both the undifferentiated and the laminated retina, coinciding in the cell body of differentiated neurons. In contrast, no correlation was found between SA-ß-GAL activity and the distribution of TUNEL-positive cells in the developing tissue.

Chromatin regulators in retinoblastoma: Biological roles and therapeutic applications.

Retinoblastoma (RB) is a pediatric ocular tumor mostly occurring due to the biallelic loss of RB1 gene in the developing retina. Early studies of genomic aberrations in RB have provided a valuable insight into how RB can progress following the tumor-initiating RB1 mutations and have established a notion that inactivation of RB1 gene is critical to initiate RB but this causative genetic lesion alone is not sufficient for malignant progression. With the advent of high-throughput sequencing technologies, we now have access to the comprehensive genomic and epigenetic landscape of RB and have come to appreciate that RB tumorigenesis requires both genetic and epigenetic alterations that might be directly or indirectly driven by RB1 loss. This integrative perspective on RB tumorigenesis has inspired research efforts to better understand the types and functions of epigenetic mechanisms contributing to RB development, leading to the identification of multiple epigenetic regulators misregulated in RB in recent years. A complete understanding of the intricate network of genetic and epigenetic factors in modulation of gene expression during RB tumorigenesis remains a major challenge but would be crucial to translate these findings into therapeutic interventions. In this review, we will provide an overview of chromatin regulators identified to be misregulated in human RB among the numerous epigenetic factors implicated in RB development. For a subset of these chromatin regulators, recent findings on their functions in RB development and potential therapeutic applications are discussed.

Development and postnatal neurogenesis in the retina: a comparison between altricial and precocial bird species.

The visual system is affected by neurodegenerative diseases caused by the degeneration of specific retinal neurons, the leading cause of irreversible blindness in humans. Throughout vertebrate phylogeny, the retina has two kinds of specialized niches of constitutive neurogenesis: the retinal progenitors located in the circumferential marginal zone and Müller glia. The proliferative activity in the retinal progenitors located in the circumferential marginal zone in precocial birds such as the chicken, the commonest bird model used in developmental and regenerative studies, is very low. This region adds only a few retinal cells to the peripheral edge of the retina during several months after hatching, but does not seem to be involved in retinal regeneration. Müller cells in the chicken retina are not proliferative under physiological conditions, but after acute damage some of them undergo a reprogramming event, dedifferentiating into retinal stem cells and generating new retinal neurons. Therefore, regenerative response after injury occurs with low efficiency in the precocial avian retina. In contrast, it has recently been shown that neurogenesis is intense in the retina of altricial birds at hatching. In particular, abundant proliferative activity is detected both in the circumferential marginal zone and in the outer half of the inner nuclear layer. Therefore, stem cell niches are very active in the retina of altricial birds. Although more extensive research is needed to assess the potential of proliferating cells in the adult retina of altricial birds, it emerges as an attractive model for studying different aspects of neurogenesis and neural regeneration in vertebrates.

A Review of DNA and Histone Methylation Alterations in the New Era of Diagnosis and Treatment of Retinal Diseases.

Epigenetics has an important role in gene regulation and other cellular processes. DNA methylation, as one of the main mechanisms of epigenetics, is a type of post-replication modifications. Aberrant DNA methylation can alter gene expression patterns; so, it plays a considerable role in the pathogenesis of many diseases. DNA methylated alterations in the promoter of specific genes can be used for the diagnosis and proprietary targets acting as a "biomarker". Early diagnosis and prevention may be possible due to these biomarkers. According to recent studies, DNA methylation abnormalities have an important role in the retinogenesis and pathogenesis of retinal diseases. Retinal diseases are the main cause of blindness and severe vision loss in the world, which will continue to increase. Also, they inflict an enormous burden on society and health care systems. Therefore, it is important to focus on the better recognition and prevention of retinal diseases and finding new targets for the treatment. DNA methylation is lionized as attractive therapeutic targets due to its reversibility. Epigenetic therapy has a high potency in the treatment of retinal diseases. Here, we reviewed the DNA and histone methylation alterations in common retinal diseases, focusing on agerelated macular degeneration (AMD), diabetic retinopathy, retinal detachment (RD), retinitis pigmentosa, retinal aging, and retinoblastoma. Then we surveyed some new approaches to epigenetic therapy in retinal disorders.

Spatio-temporal expression profile of Mettl3/Mettl14 during mouse retina development / Perfil de expresión espacio-temporal de Mettl3 / Mettl14 durante el desarrollo de la retina del ratón

SUMMARY: The Mettl3/Mettl14 methyltransferase complex installs the most ubiquitous internal mRNA modification- N6-methyladenosine (m6A). The vertebrate retina development is a multi-step process that requires fine-tuning of multiple cellular events, but very little is known about the potential function of Mettl3 and Mettl14 in this process. In this study, we demonstrated the spatio-temporal expression of Mettl3 and Mettl14 during retina development in mouse by quantitative PCR and immunofluorescence staining. We found that these two components of methyltransferase complex could be detected from the beginning of retina development; and the expression of Mettl3 and Mettl14 were gradually restricted to inner nuclear layer (INL) and ganglion cell layer (GCL); Double labeling showed that Mettl3 and Mettl14 had similar expression patterns in mature retinal INL and GCL. Overall, our spatio-temporal expression data provided the foundation for future research on the function of m6A modification in the retina development.

RESUMEN: El complejo Mettl3 / Mettl14 metiltransferasa establece la modificación interna más significativa de ARNm: N6- metiladenosina (m6A). El desarrollo de la retina de los vertebrados es un proceso de varios pasos que requiere múltiples eventos celulares; existe muy poca información sobre la función potencial de Mettl3 y Mettl14 en este proceso. En este estudio, demostramos la expresión espacio-temporal de Mettl3 y Mettl14 durante el desarrollo de la retina en ratón mediante PCR cuantitativa y tinción de inmunofluorescencia. Descubrimos que estos dos componentes del complejo de metiltransferasa podían ser detectados desde el comienzo del desarrollo de la retina; la expresión de Mettl3 y Mettl14 se restringió gradualmente a la capa nuclear interna (INL) y la capa de células ganglionares (GCL); se observó que Mettl3 y Mettl14 tenían patrones de expresión similares en INL y GCL retinianos maduros. En general, nuestros datos de expresión espacio-temporal proporcionan información para futuras investigaciones sobre la función de la modificación de m6A en el desarrollo de la retina.

Zeb2 regulates the balance between retinal interneurons and Müller glia by inhibition of BMP-Smad signaling.

The interplay between signaling molecules and transcription factors during retinal development is key to controlling the correct number of retinal cell types. Zeb2 (Sip1) is a zinc-finger multidomain transcription factor that plays multiple roles in central and peripheral nervous system development. Haploinsufficiency of ZEB2 causes Mowat-Wilson syndrome, a congenital disease characterized by intellectual disability, epilepsy and Hirschsprung disease. In the developing retina, Zeb2 is required for generation of horizontal cells and the correct number of interneurons; however, its potential function in controlling gliogenic versus neurogenic decisions remains unresolved. Here we present cellular and molecular evidence of the inhibition of Müller glia cell fate by Zeb2 in late stages of retinogenesis. Unbiased transcriptomic profiling of control and Zeb2-deficient early-postnatal retina revealed that Zeb2 functions in inhibiting Id1/2/4 and Hes1 gene expression. These neural progenitor factors normally inhibit neural differentiation and promote Müller glia cell fate. Chromatin immunoprecipitation (ChIP) supported direct regulation of Id1 by Zeb2 in the postnatal retina. Reporter assays and ChIP analyses in differentiating neural progenitors provided further evidence that Zeb2 inhibits Id1 through inhibition of Smad-mediated activation of Id1 transcription. Together, the results suggest that Zeb2 promotes the timely differentiation of retinal interneurons at least in part by repressing BMP-Smad/Notch target genes that inhibit neurogenesis. These findings show that Zeb2 integrates extrinsic cues to regulate the balance between neuronal and glial cell types in the developing murine retina.

Dystrophin Is Required for the Proper Timing in Retinal Histogenesis: A Thorough Investigation on the mdx Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal X-linked muscular disease caused by defective expression of the cytoskeletal protein dystrophin (Dp427). Selected autonomic and central neurons, including retinal neurons, express Dp427 and/or dystrophin shorter isoforms. Because of this, DMD patients may also experience different forms of cognitive impairment, neurological and autonomic disorders, and specific visual defects. DMD-related damages to the nervous system are established during development, suggesting a role for all dystrophin isoforms in neural circuit development and differentiation; however, to date, their function in retinogenesis has never been investigated. In this large-scale study, we analyzed whether the lack of Dp427 affects late retinogenesis in the mdx mouse, the most well studied animal model of DMD. Retinal gene expression and layer maturation, as well as neural cell proliferation, apoptosis, and differentiation, were evaluated in E18 and/or P0, P5, P10, and adult mice. In mdx mice, expression of Capn3, Id3 (E18-P5), and Dtnb (P5) genes, encoding proteins involved in different aspects of retina development and synaptogenesis (e.g., Calpain 3, DNA-binding protein inhibitor-3, and ß-dystrobrevin, respectively), was transiently reduced compared to age-matched wild type mice. Concomitantly, a difference in the time required for the retinal ganglion cell layer to reach appropriate thickness was observed (P0-P5). Immunolabeling for specific cell markers also evidenced a significant dysregulation in the number of GABAergic amacrine cells (P5-P10), a transient decrease in the area immunopositive for the Vesicular Glutamate Transporter 1 (VGluT1) during ribbon synapse maturation (P10) and a reduction in the number of calretinin+ retinal ganglion cells (RGCs) (adults). Finally, the number of proliferating retinal progenitor cells (P5-P10) and apoptotic cells (P10) was reduced. These results support the hypothesis of a role for Dp427 during late retinogenesis different from those proposed in consolidated neural circuits. In particular, Dp427 may be involved in shaping specific steps of retina differentiation. Notably, although most of the above described quantitative alterations recover over time, the number of calretinin+ RGCs is reduced only in the mature retina. This suggests that alterations subtler than the timing of retinal maturation may occur, a hypothesis that demands further in-depth functional studies.

MKRN2 Physically Interacts with GLE1 to Regulate mRNA Export and Zebrafish Retinal Development.

The mammalian mRNA nuclear export process is thought to terminate at the cytoplasmic face of the nuclear pore complex through ribonucleoprotein remodeling. We conduct a stringent affinity-purification mass-spectrometry-based screen of the physical interactions of human RNA-binding E3 ubiquitin ligases. The resulting protein-interaction network reveals interactions between the RNA-binding E3 ubiquitin ligase MKRN2 and GLE1, a DEAD-box helicase activator implicated in mRNA export termination. We assess MKRN2 epistasis with GLE1 in a zebrafish model. Morpholino-mediated knockdown or CRISPR/Cas9-based knockout of MKRN2 partially rescue retinal developmental defects seen upon GLE1 depletion, consistent with a functional association between GLE1 and MKRN2. Using ribonomic approaches, we show that MKRN2 binds selectively to the 3' UTR of a diverse subset of mRNAs and that nuclear export of MKRN2-associated mRNAs is enhanced upon knockdown of MKRN2. Taken together, we suggest that MKRN2 interacts with GLE1 to selectively regulate mRNA nuclear export and retinal development.

Neurogenesis and Specification of Retinal Ganglion Cells.

Across all species, retinal ganglion cells (RGCs) are the first retinal neurons generated during development, followed by the other retinal cell types. How are retinal progenitor cells (RPCs) able to produce these cell types in a specific and timely order? Here, we will review the different models of retinal neurogenesis proposed over the last decades as well as the extrinsic and intrinsic factors controlling it. We will then focus on the molecular mechanisms, especially the cascade of transcription factors that regulate, more specifically, RGC fate. We will also comment on the recent discovery that the ciliary marginal zone is a new stem cell niche in mice contributing to retinal neurogenesis, especially to the generation of ipsilateral RGCs. Furthermore, RGCs are composed of many different subtypes that are anatomically, physiologically, functionally, and molecularly defined. We will summarize the different classifications of RGC subtypes and will recapitulate the specification of some of them and describe how a genetic disease such as albinism affects neurogenesis, resulting in profound visual deficits.

Retinogenesis of the Human Fetal Retina: An Apical Polarity Perspective.

The Crumbs complex has prominent roles in the control of apical cell polarity, in the coupling of cell density sensing to downstream cell signaling pathways, and in regulating junctional structures and cell adhesion. The Crumbs complex acts as a conductor orchestrating multiple downstream signaling pathways in epithelial and neuronal tissue development. These pathways lead to the regulation of cell size, cell fate, cell self-renewal, proliferation, differentiation, migration, mitosis, and apoptosis. In retinogenesis, these are all pivotal processes with important roles for the Crumbs complex to maintain proper spatiotemporal cell processes. Loss of Crumbs function in the retina results in loss of the stratified appearance resulting in retinal degeneration and loss of visual function. In this review, we begin by discussing the physiology of vision. We continue by outlining the processes of retinogenesis and how well this is recapitulated between the human fetal retina and human embryonic stem cell (ESC) or induced pluripotent stem cell (iPSC)-derived retinal organoids. Additionally, we discuss the functionality of in utero and preterm human fetal retina and the current level of functionality as detected in human stem cell-derived organoids. We discuss the roles of apical-basal cell polarity in retinogenesis with a focus on Leber congenital amaurosis which leads to blindness shortly after birth. Finally, we discuss Crumbs homolog (CRB)-based gene augmentation.