Retinal Development Research Articles

The mechanics of the retina: Müller glia role on retinal extracellular matrix and modelling.

The retina is a highly heterogeneous tissue, both cell-wise but also regarding its extracellular matrix (ECM). The stiffness of the ECM is pivotal in retinal development and maturation and has also been associated with the onset and/or progression of numerous retinal pathologies, such as glaucoma, proliferative vitreoretinopathy (PVR), age-related macular degeneration (AMD), epiretinal membrane (ERM) formation or uveitis. Nonetheless, much remains unknown about the biomechanical milieu of the retina, and specifically the role that Müller glia play as principal mechanosensors and major producers of ECM constituents. So far, new approaches need to be developed to further the knowledge in the field of retinal mechanobiology for ECM-target applications to arise. In this review, we focus on the involvement of Müller glia in shaping and altering the retinal ECM under both physiological and pathological conditions and look into various biomaterial options to more accurately replicate the impact of matrix stiffness in vitro.

REGULATION OF RETINOGENESIS IN PRENATAL ONTOGENESIS AND ITS DISRUPTION IN AGE-RELATED MACULAR DEGENERATION

One of the main causes of blindness in the elderly in developed countries is age-related macular degeneration (AMD) [1, 2, 3]. Active research is being conducted to identify putative molecular targets for the development of an effective strategy for conservative treatment of both atrophic and neovascular forms of this disease [4, 5, 6], but at the present stage, pharmacotherapy is effective only for some patients, and is insufficient to stop the progression of the disease or eliminate the damage that has already occurred. The most promising and promising potential treatment options are considered to be the introduction of cellular technologies with retinal cell replacement and targeted pharmacotherapy. The aim of this study was to investigate the role of the retinal development inducer, SIRT1 [7, 8, 9], in retinogenesis to justify pharmacological inhibition of this protein as a possible treatment option for neovascular AMD. Using immunohistochemistry and immunocytochemistry, we assessed the expression and subcellular localization of SIRT1 and its innate inhibitor DBC1 in the retina of fetuses, adults, and mice. We also studied SIRT1 in retinal progenitor cells derived from mice and humans, the former having been used in small interfering RNA studies. SIRT1 is widely expressed in the developing and adult retina and is a regulator of key genes in retinal development, namely PAX6, Nestin, and CRX [10, 11, 12]. Moreover, we found that photoreceptor progenitor cells were among the smallest cells in a heterogeneous mouse retinal progenitor cell population [13, 14, 15]. Collectively, these results provide a basis for manipulating SIRT1 expression in small retinal progenitors as a means to increase the yield of photoreceptors for transplantation in retinal degeneration models [16, 17]. Furthermore, SIRT1 was found to be highly expressed in human-derived choroidal neovascular membranes, allowing its activity to be pharmacologically inhibited by the drug nicotinamide as a potent regulator of angiogenic and hypoxic signaling in a human retinal pigment epithelial cell line at both the protein level using angiogenesis arrays and at the RNA level using whole genome microarrays [18, 19]. These results point to the SIRT1 inhibitor, Nicotinamide, as a possible agent for treatment of neovascular AMD. Further studies of Nicotinamide are warranted in animal models of AMD. To the best of our knowledge, this is the first time that a detailed analysis of SIRT1 as a regulator of both retinal development and choroidal neovascularization has been reported.

Temporal Transcriptomic Profiling of the Developing Xenopus laevis Eye.

Retinal progenitor cells (RPCs) are a multipotent and highly proliferative population that give rise to all retinal cell types during organogenesis. Defining their molecular signature is a key step towards identifying suitable approaches to treat visual impairments. Here, we performed RNA sequencing of whole eyes from Xenopus at three embryonic stages and used differential expression analysis to define the transcriptomic profiles of optic tissues containing proliferating and differentiating RPCs during retinogenesis. Gene Ontology and KEGG pathway analyses showed that genes associated with developmental pathways (including Wnt and Hedgehog signaling) were upregulated during the period of active RPC proliferation in early retinal development (Nieuwkoop Faber st. 24 and 27). Developing eyes had dynamic expression profiles and shifted to enrichment for metabolic processes and phototransduction during RPC progeny specification and differentiation (st. 35). Furthermore, conserved adult eye regeneration genes were also expressed during early retinal development, including sox2, pax6, nrl, and Notch signaling components. The eye transcriptomic profiles presented here span RPC proliferation to retinogenesis and include regrowth-competent stages. Thus, our dataset provides a rich resource to uncover molecular regulators of RPC activity and will allow future studies to address regulators of RPC proliferation during eye repair and regrowth.

Chrdl1-mediated BMP4 inhibition disrupts the balance between retinal neurons and Müller Glia

Chordin-like 1 (CHRDL1) is a secreted protein that serves as an endogenous antagonist of bone morphogenetic proteins (BMPs). In the developing retina, Bmp4 has been demonstrated to be essential for sustaining the proliferation of progenitor cells and facilitating the differentiation of glial cells. Despite these efforts, the precise effects of Bmp4 inhibition on the developing retina are yet to be fully understood. We sought to address this question by overexpressing Chrdl1 in the developing retina. In this study, we explored the impact of Bmp4 inhibition on the developing mouse retina by conditionally overexpressing the Bmp4 inhibitor Chrdl1. Initially, we characterized the expression patterns of Bmp4 and Chrdl1 in the developing mouse retina from E10.5 to P12.5. Additionally, we utilized various molecular markers to demonstrate that Bmp4 inhibition disrupts both neuronal and Müller glial differentiation in the developing mouse retina. Moreover, through the application of RNA-seq analysis, distinctively expressed retinal genes under the modulation of Bmp4 signaling were discerned, encompassing the upregulation of Id1/2/3/4 and Hes1/5, as well as the downregulation of Neurod1/2/4 and Bhlhe22/23. Lastly, electroretinogram (ERG) and optomotor response (OMR) assays were conducted to illustrate that Bmp4 inhibition impairs the functional connectivity of various cells in the retina and consequently affects visual function. Collectively, this study demonstrates that inhibiting Bmp4 promotes the differentiation of retinal neurons over Müller glia by activating the expression of genes associated with neuron specification. These findings offer molecular insights into the role of Bmp4 signaling in mammalian retinal development.

Single cell dual-omic atlas of the human developing retina

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.

Generation of an Armcx1 Conditional Knockout Mouse.

Armadillo repeat-containing X-linked protein-1 (Armcx1) is a poorly characterized transmembrane protein that regulates mitochondrial transport in neurons. Its overexpression has been shown to induce neurite outgrowth in embryonic neurons and to promote retinal ganglion cell (RGC) survival and axonal regrowth in a mouse optic nerve crush model. In order to evaluate the functions of endogenous Armcx1 in vivo, we have created a conditional Armcx1 knockout mouse line in which the entire coding region of the Armcx1 gene is flanked by loxP sites. This Armcx1fl line was crossed with mouse strains in which Cre recombinase expression is driven by the promoters for β-actin and Six3, in order to achieve deletion of Armcx1 globally and in retinal neurons, respectively. Having confirmed deletion of the gene, we proceeded to characterize the abundance and morphology of RGCs in Armcx1 knockout mice aged to 15 months. Under normal physiological conditions, no evidence of aberrant retinal or optic nerve development or RGC degeneration was observed in these mice. The Armcx1fl mouse should be valuable for future studies investigating mitochondrial morphology and transport in the absence of Armcx1 and in determining the susceptibility of Armcx1-deficient neurons to degeneration in the setting of additional heritable or environmental stressors.

M2 macrophage-derived extracellular vesicles ameliorate Benzalkonium Chloride-induced dry eye

Dry eye disease (DED) is a common ocular condition affecting a significant portion of the global population, yet effective treatment options remain elusive. This study investigates the therapeutic potential of M2 macrophage-derived extracellular vesicles (M2-EVs) in a mouse model of DED. The DED model was established using 0.2% benzalkonium chloride (BAC) eye drops, applied twice daily for a week. Post induction, the mice were categorized into 5 groups: PBS, Sodium Hyaluronate (HA, 0.1%), Fluoromethalone (FM, 0.1%), M0-EVs, and M2-EVs. The efficacy of M2-EVs was assessed through tear production, corneal fluorescein staining and HE staining. RNA sequencing (RNA-seq) was employed to investigate the mechanisms underlying the therapeutic effects of M2-EVs in DED. Notably, the M2-EVs treated group exhibited the highest tear secretion, indicating improved tear film stability and reduced corneal surface damage. Histological analysis revealed better corneal structure organization in the M2-EVs group, suggesting enhanced ocular surface repair and corneal preservation. Furthermore, M2-EVs treatment significantly decreased pro-inflammatory cytokine levels and showed unique enrichment of genes related to retinal development. These findings suggest that M2-EVs could serve as a promising noninvasive therapeutic approach for human DED, targeting ocular surface inflammation.

Lack of FMRP in the retina: Evidence of a retinal specific transcriptomic profile

Fragile X Syndrome (FXS), the most common inherited form of human intellectual disability, is a monogenic neurodevelopmental disorder caused by a loss-of-function mutation of the FMR1 gene. FMR1 is encoding the Fragile X Messenger Ribonucleo Protein (FMRP) an RNA-binding protein that regulates the translation of synaptic proteins. The absence of FMRP expression has many important consequences on synaptic plasticity and function, leading to the FXS clinical phenotype. Over the last decade, a visual neurosensorial phenotype had been described in the FXS patients as well as in the murine model (Fmr1-/ymice), characterized by retinal deficits associated to retinal perception alterations. However, although the transcriptomic profile in the absence of FMRP has been studied in the cerebral part of the central nervous system (CNS), there are no actual data for the retina which is an extension of the CNS. Herein, we investigate the transcriptomic profile of mRNA from whole retinas of Fmr1-/ymice. Interestingly, we found a specific signature of Fmrp absence on retinal mRNA expression with few common genes compared to other brain studies. Gene Ontology on these retinal specific genes demonstrated an enrichment in retinal development genes as well as in synaptic genes. These alterations could be linked to the reported retinal phenotype of the FXS condition. In conclusion, we describe for the first time, retinal-specific transcriptomic changes in the absence of FMRP.

Human microglia-derived proinflammatory cytokines facilitate human retinal ganglion cell development and regeneration

Microglia (μG), the resident immune cells in the central nervous system, surveil the parenchyma to maintain the structural and functional homeostasis of neurons. Besides, they influence neurogenesis and synaptogenesis through complement-mediated phagocytosis. Emerging evidence suggests that μG may also influence development through proinflammatory cytokines. Here, we examined the premise that tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β), the two most prominent components of the μG secretome, influence retinal development, specifically the morphological and functional differentiation of human retinal ganglion cells (hRGCs). Using controlled generation of hRGCs and human μG (hμG) from pluripotent stem cells, we demonstrate that TNF-α and IL-1β secreted by unchallenged hμG did not influence hRGC generation. However, their presence significantly facilitated neuritogenesis along with the basal function of hRGCs, which involved the recruitment of the AKT/mTOR pathway. We present exvivo evidence that proinflammatory cytokines may play an important role in the morphological and physiological maturation of hRGCs, which may be recapitulated for regeneration.

Development of Non-Invasive miRNA Markers for Assessing the Quality of Human Induced Pluripotent Stem Cell-Derived Retinal Organoids.

Human retinal organoids (ROs) have emerged as valuable tools for studying retinal development, modeling human retinal diseases, and screening drugs. However, their application is limited primarily due to time-intensive generation, high costs, and low reproducibility. Quality assessment of RO differentiation is crucial for their application in research. However, traditional methods such as morphological evaluation and immunohistochemical analysis have limitations due to their lack of precision and invasiveness, respectively. This study aims to identify non-invasive biomarkers for RO differentiation quality using exosomal microRNAs (miRNAs), which are known to reflect cell-specific functions and development in the retina. We differentiated ROs from human induced pluripotent stem cells (hiPSCs) and classified them into 'superior' and 'inferior' groups based on morphological and immunohistochemical criteria. Exosomes from the conditioned media were isolated and analyzed for miRNA content. Our findings revealed distinct miRNA profiles between superior and inferior ROs, with superior ROs exhibiting higher miRNA diversity and specifically up- or down-regulated miRNAs. Gene ontology and pathway enrichment analyses indicated that the target genes of these miRNAs are involved in neuron proliferation and differentiation. The study suggests the potential of exosomal hsa-miR-654-3p and hsa-miR-451a as non-invasive biomarkers for real-time monitoring of RO quality, facilitating the development of standardized, efficient, and cost-effective culture methods.

Temporal Transcriptomic Profiling of the Developing Xenopus laevis Eye.

Retinal progenitor cells (RPCs) are a multipotent and highly proliferative population that give rise to all retinal cell types during organogenesis. Defining their molecular signature is a key step towards identifying suitable approaches to treat visual impairments. Here, we performed RNA-sequencing of whole eyes from Xenopus at three embryonic stages and used differential expression analysis to define the transcriptomic profiles of optic tissues containing proliferating and differentiating RPCs during retinogenesis. Gene Ontology and KEGG pathway analyses showed that genes associated with developmental pathways (including Wnt and Hedgehog signaling) were upregulated during the period of active RPC proliferation in early retinal development (Nieuwkoop Faber st. 24 and 27). Developing eyes had dynamic expression profiles and shifted to enrichment for metabolic processes and phototransduction during RPC progeny specification and differentiation (st. 35). Furthermore, conserved adult eye regeneration genes were also expressed during early retinal development including sox2, pax6, nrl, and Notch signaling components. The eye transcriptomic profiles presented here span RPC proliferation to retinogenesis and included regrowth-competent stages. Thus, our dataset provides a rich resource to uncover molecular regulators of RPC activity and will allow future studies to address regulators of RPC proliferation during eye repair and regrowth.

Early-life exposure to five biodegradable plastics impairs eye development and visually-mediated behavior through disturbing hypothalamus-pituitary-thyroid (HPT) axis in zebrafish larvae

Biodegradable plastics have been commonly developed and applied as an alternative to traditional plastics, which cause environmental plastic pollution. However, biodegradable plastics still present limitations such as stringent degradation conditions and slow degradation rate, and may cause harm to the environment and organisms. Consequently, in this study, zebrafish was used to evaluate the effects of five biodegradable microplastics (MPs), polyglycolic acid (PGA), polylactic acid (PLA), polybutylene succinate (PBS), polyhydroxyalkanoate (PHA) and polybutylene adipate terephthalate (PBAT) exposure on the early development, retina morphology, visually-mediated behavior, and thyroid signaling at concentrations of 1 mg/L and 100 mg/L. The results indicated that all MPs induced decreased survival rate, reduced body length, smaller eyes, and smaller heads, affecting the early development of zebrafish larvae. Moreover, the thickness of retinal layers, including inner plexiform layer (IPL), outer nuclear layer (ONL), and retinal ganglion layer (RGL) was decreased, and the expression of key genes related to eye and retinal development was abnormally altered after all MPs exposure. Exposure to PBS and PBAT led to abnormal visually-mediated behavior, indicating likely affected the visual function. All MPs could also cause thyroid system disorders, among which alterations in the thyroid hormone receptors (TRs) genes could affect the retinal development of zebrafish larvae. In summary, biodegradable MPs exhibited eye developmental toxicity and likely impaired the visual function in zebrafish larvae. This provided new evidence for revealing the effects of biodegradable plastics on aquatic organism development and environmental risks to aquatic ecosystems.

PHLDA1-PRDM1 mediates the effect of lentiviral vectors on fate-determination of human retinal progenitor cells

Lentiviral vectors have markedly enhanced gene therapy efficiency in treating congenital diseases, but their long-term safety remains controversial. Most gene therapies for congenital eye diseases need to be carried out at early ages, yet the assessment of related risks to ocular development posed by lentiviral vectors is challenging. Utilizing single-cell transcriptomic profiling on human retinal organoids, this study explored the impact of lentiviral vectors on the retinal development and found that lentiviral vectors can cause retinal precursor cells to shift toward photoreceptor fate through the up-regulation of key fate-determining genes such as PRDM1. Further investigation demonstrated that the intron and intergenic region of PRDM1 was bound by PHLDA1, which was also up-regulated by lentiviral vectors exposure. Importantly, knockdown of PHLDA1 successfully suppressed the lentivirus-induced differentiation bias of photoreceptor cells. The findings also suggest that while lentiviral vectors may disrupt the fate determination of retinal precursor cells, posing risks in early-stage retinal gene therapy, these risks could potentially be reduced by inhibiting the PHLDA1-PRDM1 axis.

The intracellular domain of Sema6A is essential for development of the zebrafish retina.

Semaphorin6A (Sema6A) is a repulsive guidance molecule that plays many roles in central nervous system, heart and bone development, as well as immune system responses and cell signaling in cancer. Loss of Sema6A or its receptor PlexinA2 in zebrafish leads to smaller eyes and improper retinal patterning. Here, we investigate a potential role for the Sema6A intracellular domain in zebrafish eye development and dissect which phenotypes rely on forward signaling and which rely on reverse signaling. We performed rescue experiments on zebrafish Sema6A morphants with either full-length Sema6A (Sema6A-FL) or Sema6A lacking its intracellular domain (Sema6A-ΔC). We identified that the intracellular domain is not required for eye size and retinal patterning, however it is required for retinal integrity, the number and end feet strength of Müller glia and protecting against retinal cell death. This novel function for the intracellular domain suggests a role for Sema6A reverse signaling in zebrafish eye development.

Characteristics of retinal vascularization in reactivated retinopathy of prematurity requiring treatment and clinical outcome after reinjection of ranibizumab

This study aimed to determine whether the state of retinal vascularization after anti-vascular endothelial growth factor (anti-VEGF) injection can help predict the risk of reactivated retinopathy of prematurity (ROP) requiring treatment and whether repeated ranibizumab injection will be effective in such cases. We retrospectively reviewed 24 infants (43 eyes) who received ranibizumab monotherapy between January 2021 and December 2022. All eyes were classified as having non-retreated ROP or retreated ROP. The state of ROP at the time of treatment, the time required for resolution of plus disease, and the extent of vascularization at 4 and 8 weeks after treatment were analyzed. Extent of temporal retinal vascularization was measured with serial fundus images using disc-fovea distance (DF) unit and disc diameter (DD). Reactivated ROP requiring treatment occurred in six infants (25.0%) and ten eyes (23.3%) after ranibizumab treatment. The mean retreatment interval was 9.0 ± 3.3 weeks (range 4–16). In the retreated ROP group, the time required for the resolution of plus disease after primary injection was longer compared to the control group (13.3 days vs 5.2 days), with a mean ROP regression time of 3.4 weeks. All eyes in the retreated ROP showed retinal vascularization < 0.5 DF from the original site at 4 weeks after injection. In 90% of cases with retreated ROP, the extent of vascularization at 8 weeks after injection was within 1 DF from the original ROP site, and all cases showed reactivation in the posterior Zone II area. The extent of retinal neovascularization in the retreated group was an average of 0.7 DD (vs 1.7 DD) and 1.3 DD (vs 3.3 DD) at 4 and 8 weeks after injection, respectively. After ranibizumab retreatment, only one reactivated case with vitreous traction progressed to focal retinal detachment, while all other cases regressed with peripheral vascular development. The continuation of delayed retinal blood vessel development after ≥ 8 weeks may indicate a high likelihood of reactivated ROP requiring treatment. In the absence of vitreous traction, ranibizumab reinjection is likely to be effective in treating reactivated ROP requiring treatment.

Thyroid Hormone Signaling in Retinal Development and Function: Implications for Diabetic Retinopathy and Age-Related Macular Degeneration.

Thyroid Hormones (THs) play a central role in the development, cell growth, differentiation, and metabolic homeostasis of neurosensory systems, including the retina. The coordinated activity of various components of TH signaling, such as TH receptors (THRs) and the TH processing enzymes deiodinases 2 and 3 (DIO2, DIO3), is required for proper retinal maturation and function of the adult photoreceptors, Müller glial cells, and pigmented epithelial cells. Alterations of TH homeostasis, as observed both in frank or subclinical thyroid disorders, have been associated with sight-threatening diseases leading to irreversible vision loss i.e., diabetic retinopathy (DR), and age-related macular degeneration (AMD). Although observational studies do not allow causal inference, emerging data from preclinical models suggest a possible correlation between TH signaling imbalance and the development of retina disease. In this review, we analyze the most important features of TH signaling relevant to retinal development and function and its possible implication in DR and AMD etiology. A better understanding of TH pathways in these pathological settings might help identify novel targets and therapeutic strategies for the prevention and management of retinal disease.

Electrophysiological Analysis of Retinal Organoid Development Using 3D Microelectrodes of Liquid Metals.

Despite of the substantial potential of human-derived retinal organoids, the degeneration of retinal ganglion cells (RGCs) during maturation limits their utility in assessing the functionality of later-born retinal cell subtypes. Additionally, conventional analyses primarily rely on fluorescent emissions, which limits the detection of actual cell functionality while risking damage to the 3D cytoarchitecture of organoids. Here, an electrophysiological analysis is presented to monitor RGC development in early to mid-stage retinal organoids, and compare distinct features with fully-mature mouse retina. This approach utilizes high-resolution 3D printing of liquid-metal microelectrodes, enabling precise targeting of specific inner retinal layers within organoids. The adaptable distribution and softness of these microelectrodes facilitate the spatiotemporal recording of inner retinal signals. This study not only demonstrates the functional properties of RGCs in retinal organoid development but also provides insights into their synaptic connectivity, reminiscent of fetal native retinas. Further comparison with fully-mature mouse retina in vivo verifies the organoid features, highlighting the potential of early-stage retinal organoids in biomedical research.

Identification and Characterization of ATOH7-Regulated Target Genes and Pathways in Human Neuroretinal Development.

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

Endothelial Zmiz1 modulates physiological and pathophysiological angiogenesis during retinal development.

Angiogenesis is a highly coordinated process involving the control of various endothelial cell behaviors. Mechanisms for transcription factor involvement in the regulation of endothelial cell dynamics and angiogenesis have become better understood, however much remains unknown, especially the role of non-DNA binding transcriptional cofactors. Here, we show that Zmiz1, a transcription cofactor, is enriched in the endothelium and critical for embryonic vascular development, postnatal retinal angiogenesis, and pathological angiogenesis in oxygen induced retinopathy (OIR). In mice, endothelial cell-specific deletion of Zmiz1 during embryogenesis led to lethality due to abnormal angiogenesis and vascular defects. Inducible endothelial cell-specific ablation of Zmiz1 postnatally resulted in impaired retinal vascular outgrowth, decreased vascular density, and increased vessel regression. In addition, angiogenic sprouting in the superficial and deep layers of the retina was markedly reduced. Correspondingly, vascular sprouting in fibrin bead assays was significantly reduced in the absence of Zmiz1, while further in vitro and in vivo evidence also suggested deficits in EC migration. In agreement with the defective sprouting angiogenesis phenotype, gene expression analysis of isolated retinal endothelial cells revealed downregulation of tip-cell enriched genes upon inactivation of Zmiz1. Lastly, our study suggested that endothelial Zmiz1 is critical for intraretinal revascularization following hypoxia exposure in the OIR model. Taken together, these findings begin to define the previously unspecified role of endothelial Zmiz1 in physiological and pathological angiogenesis.

Evolutionary conservation of VSX2 super-enhancer modules in retinal development.

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