Developing Mouse Retina Research Articles

Identification of a non-canonical planar cell polarity pathway triggered by light in the developing mouse retina.

The coordinated spatial arrangement of organelles within a tissue plane, known as planar cell polarity (PCP), is critical for organ development and function. Gradients of morphogens and their receptors typically set-up PCP, but whether non-molecular cues, akin to phototropism in plants, also play a part remains unknown. Here, we report that basal bodies of newborn photoreceptor cells in the mouse retina are positioned centrally on the apical surface but then move laterally during the first postnatal week, generating cell-intrinsic asymmetry in the retinal plane. After 1week, when the eyes open, basal bodies of cone cilia, but not rods, become coordinated across the plane to face the center of the retina. We further show that light is essential for cone PCP, triggering a cascade in which cone transducin interacts with the G-protein-signaling modulator protein 2 (GPSM2) to establish PCP. This work identifies a non-canonical PCP pathway initiated by light.

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.

Notch signaling regulates UNC5B to suppress endothelial proliferation, migration, junction activity, and retinal plexus branching

Notch signaling guides vascular development and function by regulating diverse endothelial cell behaviors, including migration, proliferation, vascular density, endothelial junctions, and polarization in response to flow. Notch proteins form transcriptional activation complexes that regulate endothelial gene expression, but few of the downstream effectors that enable these phenotypic changes have been characterized in endothelial cells, limiting our understanding of vascular Notch activities. Using an unbiased screen of translated mRNA rapidly regulated by Notch signaling, we identified novel in vivo targets of Notch signaling in neonatal mouse brain endothelium, including UNC5B, a member of the netrin family of angiogenic-regulatory receptors. Endothelial Notch signaling rapidly upregulates UNC5B in multiple endothelial cell types. Loss or gain of UNC5B recapitulated specific Notch-regulated phenotypes. UNC5B expression inhibited endothelial migration and proliferation and was required for stabilization of endothelial junctions in response to shear stress. Loss of UNC5B partially or wholly blocked the ability of Notch activation to regulate these endothelial cell behaviors. In the developing mouse retina, endothelial-specific loss of UNC5B led to excessive vascularization, including increased vascular outgrowth, density, and branchpoint count. These data indicate that Notch signaling upregulates UNC5B as an effector protein to control specific endothelial cell behaviors and inhibit angiogenic growth.

Non-canonical Wnt pathway expression in the developing mouse and human retina

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.

Temporal landscape and translational regulation of A-to-I RNA editing in mouse retina development

BackgroundThe significance of A-to-I RNA editing in nervous system development is widely recognized; however, its influence on retina development remains to be thoroughly understood.ResultsIn this study, we performed RNA sequencing and ribosome profiling experiments on developing mouse retinas to characterize the temporal landscape of A-to-I editing. Our findings revealed temporal changes in A-to-I editing, with distinct editing patterns observed across different developmental stages. Further analysis showed the interplay between A-to-I editing and alternative splicing, with A-to-I editing influencing splicing efficiency and the quantity of splicing events. A-to-I editing held the potential to enhance translation diversity, but this came at the expense of reduced translational efficiency. When coupled with splicing, it could produce a coordinated effect on gene translation.ConclusionsOverall, this study presents a temporally resolved atlas of A-to-I editing, connecting its changes with the impact on alternative splicing and gene translation in retina development.

G protein-coupled estrogen receptor expression in postnatal developing mouse retina.

Estrogen has emerged as a multifaceted signaling molecule in the retina, playing an important role in neural development and providing neuroprotection in adults. It interacts with two receptor types: classical estrogen receptors (ERs) alpha and beta, and G protein-coupled estrogen receptor (Gper). Gper differs from classical ERs in structure, localization, and signaling. Here we provide the first report of the temporal and spatial properties of Gper transcript and protein expression in the developing and mature mouse retina. We applied qRT-PCR to determine Gper transcript expression in wild type mouse retina from P0-P21. Immunohistochemistry and Western blot were used to determine Gper protein expression and localization at the same time points. Gper expression showed a 6-fold increase during postnatal development, peaking at P14. Relative total Gper expression exhibited a significant decrease during retinal development, although variations emerged in the timing of changes among different forms of the protein. Gper immunoreactivity was seen in retinal ganglion cells (RGCs) throughout development and also in somas in the position of horizontal cells at early time points. Immunoreactivity was observed in the cytoplasm and Golgi at all time points, in the nucleus at early time points, and in RGC axons as the retina matured. In conclusion, our study illuminates the spatial and temporal expression patterns of Gper in the developing mouse retina and provides a vital foundation for further investigations into the role of Gper in retinal development and degeneration.

Label-aware distance mitigates temporal and spatial variability for clustering and visualization of single-cell gene expression data

Clustering and visualization are essential parts of single-cell gene expression data analysis. The Euclidean distance used in most distance-based methods is not optimal. The batch effect, i.e., the variability among samples gathered from different times, tissues, and patients, introduces large between-group distance and obscures the true identities of cells. To solve this problem, we introduce Label-Aware Distance (Lad), a metric using temporal/spatial locality of the batch effect to control for such factors. We validate Lad on simulated data as well as apply it to a mouse retina development dataset and a lung dataset. We also found the utility of our approach in understanding the progression of the Coronavirus Disease 2019 (COVID-19). Lad provides better cell embedding than state-of-the-art batch correction methods on longitudinal datasets. It can be used in distance-based clustering and visualization methods to combine the power of multiple samples to help make biological findings.

Light activates Ube3a, an Angelman syndrome-associated gene, by mediating the chromatin structures during postnatal development of mouse retina.

Angelman syndrome, a severe neurodevelopmental disorder, is primarily caused by mutations or deletions of maternally inherited ubiquitin protein ligase E3A (UBE3A). Activation of the silenced paternal copy of UBE3A can occur with pharmacological perturbation; however, an environmental approach has not been examined. Here, we found Ube3a is highly expressed in embryonic and early neonatal mouse retina and is maternally-, but not paternally-, expressed in ganglion cells, amacrine cells, and horizontal cells. Moreover, we analyzed UBE3A expression in the retina and visual cortex of postnatal day 28 mice (P28) following exposure to light emissions from white compact-fluorescent bulbs or blue light-emitting diodes from postnatal day 0 (P0) to 28 (P28), encompassing a crucial phase of visual system development. We found higher levels of Ube3a RNA and protein in the retina, but not visual cortex compared with tissues from P28 mice exposure to typical lighting (controls). Levels of both paternal- and maternal-UBE3A protein in mouse retina were higher than controls in P28 mice exposed to white or blue light. Moreover, levels of open and repressive chromatin structures, indicated by histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3), respectively, were increased in the Ube3a promoter from mouse retina exposed to white or blue light. Our findings strongly suggest that extended exposure to white or blue light constitutes a substantial environmental factor that can effectively promote UBE3A expression within the central nervous system.

The landscape of the long non-coding RNAs in developing mouse retinas

BackgroundThe long non-coding RNAs (lncRNAs) are critical regulators of diverse biological processes. Nevertheless, a global view of its expression and function in the mouse retina, a crucial model for neurogenesis study, still needs to be made available.ResultsHerein, by integrating the established gene models and the result from ab initio prediction using short- and long-read sequencing, we characterized 4,523 lncRNA genes (MRLGs) in developing mouse retinas (from the embryonic day of 12.5 to the neonatal day of P28), which was so far the most comprehensive collection of retinal lncRNAs. Next, derived from transcriptomics analyses of different tissues and developing retinas, we found that the MRLGs were highly spatiotemporal specific in expression and played essential roles in regulating the genesis and function of mouse retinas. In addition, we investigated the expression of MRLGs in some mouse mutants and revealed that 97 intergenic MRLGs might be involved in regulating differentiation and development of retinal neurons through Math5, Isl1, Brn3b, NRL, Onecut1, or Onecut2 mediated pathways.ConclusionsIn summary, this work significantly enhanced our knowledge of lncRNA genes in mouse retina development and provided valuable clues for future exploration of their biological roles.

CEP162 deficiency causes human retinal degeneration and reveals a dual role in ciliogenesis and neurogenesis.

Defects in primary or motile cilia result in a variety of human pathologies, and retinal degeneration is frequently associated with these so-called ciliopathies. We found that homozygosity for a truncating variant in CEP162, a centrosome and microtubule-associated protein required for transition zone assembly during ciliogenesis and neuronal differentiation in the retina, caused late-onset retinitis pigmentosa in 2 unrelated families. The mutant CEP162-E646R*5 protein was expressed and properly localized to the mitotic spindle, but it was missing from the basal body in primary and photoreceptor cilia. This impaired recruitment of transition zone components to the basal body and corresponded to complete loss of CEP162 function at the ciliary compartment, reflected by delayed formation of dysmorphic cilia. In contrast, shRNA knockdown of Cep162 in the developing mouse retina increased cell death, which was rescued by expression of CEP162-E646R*5, indicating that the mutant retains its role for retinal neurogenesis. Human retinal degeneration thus resulted from specific loss of the ciliary function of CEP162.

Exosomes incorporated with black phosphorus quantum dots attenuate retinal angiogenesis via disrupting glucose metabolism.

Black phosphorus quantum dots (BPQDs) have shown potential in tumor therapy, however, their anti-angiogenic functions have not been studied. Although BPQDs are easily degraded to non-toxic phosphrous, the reported toxicity, poor stability, and non-selectivity largely limit their further application in medicine. In this study, a vascular targeting, biocompatible, and cell metabolism-disrupting nanoplatform is engineered by incorporating BPQDs into exosomes modified with the Arg-Gly-Asp (RGD) peptide (BPQDs@RGD-EXO nanospheres, BREs). BREs inhibit endothelial cells (ECs) proliferation, migration, tube formation, and sprouting in vitro. The anti-angiogenic role of BREs in vivo is evaluated using mouse retinal vascular development model and oxygen-induced retinopathy model. Combined RNA-seq and metabolomic analysis reveal that BREs disrupt glucose metabolism, which is further confirmed by evaluating metabolites, ATP production and the c-MYC/Hexokinase 2 pathway. These BREs are promising anti-angiogenic platforms for the treatment of pathological retinal angiogenesis with minimal side effects.

Ikaros family proteins redundantly regulate temporal patterning in the developing mouse retina.

Temporal identity factors regulate competence of neural progenitors to generate specific cell types in a time-dependent manner, but how they operate remains poorly defined. In the developing mouse retina, the Ikaros zinc-finger transcription factor Ikzf1 regulates production of early-born cell types, except cone photoreceptors. In this study we show that, during early stages of retinal development, another Ikaros family protein, Ikzf4, functions redundantly with Ikzf1 to regulate cone photoreceptor production. Using CUT&RUN and functional assays, we show that Ikzf4 binds and represses genes involved in late-born rod photoreceptor specification, hence favoring cone production. At late stages, when Ikzf1 is no longer expressed in progenitors, we show that Ikzf4 re-localizes to target genes involved in gliogenesis and is required for Müller glia production. We report that Ikzf4 regulates Notch signaling genes and is sufficient to activate the Hes1 promoter through two Ikzf GGAA-binding motifs, suggesting a mechanism by which Ikzf4 may influence gliogenesis. These results uncover a combinatorial role for Ikaros family members during nervous system development and provide mechanistic insights on how they temporally regulate cell fate output.

Cystathionine-γ-lyase overexpression modulates oxidized nicotinamide adenine dinucleotide biosynthesis and enhances neovascularization

ObjectiveHydrogen sulfide is a proangiogenic gas produced primarily by the transsulfuration enzyme cystathionine-γ-lyase (CGL). CGL-dependent hydrogen sulfide production is required for neovascularization in models of peripheral arterial disease. However, the benefits of increasing endogenous CGL and its mechanism of action have not yet been elucidated. MethodsMale whole body CGL-overexpressing transgenic (CGLTg) mice and wild-type (WT) littermates (C57BL/6J) were subjected to the hindlimb ischemia model (age, 10-12 weeks). Functional recovery was assessed via the treadmill exercise endurance test. Leg perfusion was measured by laser Doppler imaging and vascular endothelial-cadherin immunostaining. To examine the angiogenic potential, aortic ring sprouting assay and postnatal mouse retinal vasculature development studies were performed. Finally, comparative metabolomics analysis, oxidized/reduced nicotinamide adenine dinucleotide (NAD+/NADH) analysis, and quantitative real-time polymerase chain reaction were performed on CGLWT and CGLTg gastrocnemius muscle. ResultsThe restoration of blood flow occurred more rapidly in CGLTg mice. Compared with the CGLWT mice, the median ± standard deviation running distance and time were increased for the CGLTg mice after femoral artery ligation (159 ± 53 m vs 291 ± 74 m [P < .005] and 17 ± 4 minutes vs 27 ± 5 minutes [P < .05], respectively). Consistently, in the CGLTg ischemic gastrocnemius muscle, the capillary density was increased fourfold (0.05 ± 0.02 vs 0.20 ± 0.12; P < .005). Ex vivo, the endothelial cell (EC) sprouting length was increased in aorta isolated from CGLTg mice, especially when cultured in VEGFA (vascular endothelial growth factor A)-only media (63 ± 2 pixels vs 146 ± 52 pixels; P < .05). Metabolomics analysis demonstrated a higher level of niacinamide, a precursor of NAD+/NADH in the muscle of CGLTg mice (61.4 × 106 ± 5.9 × 106 vs 72.4 ± 7.7 × 106 area under the curve; P < .05). Similarly, the NAD+ salvage pathway gene expression was increased in CGLTg gastrocnemius muscle. Finally, CGL overexpression or supplementation with the NAD+ precursor nicotinamide mononucleotide improved EC migration in vitro (wound closure: control, 35% ± 9%; CGL, 55% ± 11%; nicotinamide mononucleotide, 42% ± 13%; P < .05). ConclusionsOur results have demonstrated that CGL overexpression improves the neovascularization of skeletal muscle on hindlimb ischemia. These effects correlated with changes in the NAD pathway, which improved EC migration.

MODL-30. AN ENGINEERED TRANSGENIC MOUSE MODEL OF RETINOBLASTOMA

Abstract Retinoblastoma, the most common intraocular tumor of childhood, was recently shown to comprise two major molecular subtypes. Subtype 1 includes most heritable forms, with germline mutation of RB1 and a cone-differentiated state. Subtype 2 is often non-hereditary, exhibits stemness features, increased expression of MYCN and its target genes, and a high risk of metastasis. Relative to subtype 1, subtype 2 tumors have a worse prognosis. To date, no mouse model has been available for studying subtype 2 disease. It is known that SHH pathway proteins including SHH, GLI1 and GLI2 are abnormally elevated in human retinoblastoma. We recently discovered that activation of GLI2 in the developing mouse retina leads to intraocular tumors that exhibit cellular and molecular features resembling human subtype 2 retinoblastoma with aberrant upregulation of MYCN and its target genes. Because MYCN is a downstream target of the SHH pathway, we hypothesize that inhibiting the SHH-GLI2-MYCN signaling axis may effectively treat subtype 2 disease. Using our GLI2 based mouse model of retinoblastoma, we will elucidate mechanisms underlying tumor initiation and progression, and evaluate the therapeutic potential of SHH and GLI2 inhibitors in preventing retinoblastoma. We will validate our mouse model findings using human retinoblastoma cell lines and patient-derived tumor xenografts. Our studies will yield new mechanistic insights into the development of subtype 2 retinoblastoma and identify novel molecular targets for early detection, treatment, and prevention of ocular tumors and associated metastatic disease.

Brca1 Is Regulated by the Transcription Factor Gata3, and Its Silencing Promotes Neural Differentiation in Retinal Neurons.

Previous studies have indicated that Brca1 (Breast cancer suppressor gene 1) plays an important role in neural development and degenerative diseases. However, the bioactivity and regulatory mechanism of Brca1 expression in retinal neurocytes remain unclear. In the present study, our data indicated that Brca1 maintains the state of neuronal precursor cells. Brca1 silencing induces differentiation in 661W cells. Nestin, a marker of precursor cells, was significantly decreased in parallel with Brca1 silencing in 661W cells, whereas Map2 (Microtubule associated protein 2), a marker of differentiated neurons, was significantly increased. Neurite outgrowth was increased by ~4.0-fold in Brca1-silenced cells. Moreover, DNA affinity purification assays and ChIP assays demonstrated that Gata3 (GATA binding protein 3) regulates Brca1 transcription in 661W cells. Silencing or overexpressing Gata3 could significantly regulate the expression of Brca1 and affect its promoter inducibility. Furthermore, the expression of Gata3 generally occurred in parallel with that of Brca1 in developing mouse retinas. Both Gata3 and Brca1 are expressed in the neonatal mouse retina but are developmentally silenced with age. Exogenous Gata3 significantly inhibited neural activity by decreasing synaptophysin and neurite outgrowth. Thus, this study demonstrated that Brca1 is transcriptionally regulated by Gata3. Brca1/Gata3 silencing is involved in neuronal differentiation and maturation.

Loss-of-function approach using mouse retinal explants showed pivotal roles of Nmnat2 in early and middle stages of retinal development.

Nicotinamide mononucleotide adenylyltransferase (Nmnat) is a class of enzymes with three members (Nmnat1-3). Nmnat1 is in nucleus and associated with Leber congenital amaurosis, a form of early-onset retinal degeneration, while Nmnat2 is in cytoplasm and a well-characterized neuroprotective factor. The differences in their biological roles in the retina are unclear. We performed short hairpin RNA (shRNA)-based loss-of-function analysis of Nmnat2 during mouse retinal development in retinal explant cultures prepared from early (E14.5), middle (E17.5), or late (postnatal day [P]0.5) developmental stages. Nmnat2 has important roles in the survival of retinal cells in the early and middle stages of retinal development. Retinal cell death caused by Nmnat2 knockdown could be partially rescued by supplementation with NAD or nicotinamide mononucleotide (NMN). Survival of retinal cells in the late stage of retinal development was unaffected by Nmnat2, but differentiation of Müller glia was controlled by Nmnat2. RNA-Seq analyses showed perturbation of gene expression patterns by shRNAs specific for Nmnat1 or Nmnat2, but gene ontology analysis did not provide a rational explanation for the phenotype. This study showed that Nmnat2 has multiple developmental stage-dependent roles during mouse retinal development, which were clearly different from those of Nmnat1, suggesting specific roles for Nmnat1 and Nmnat2.

Developmental genome-wide occupancy analysis of bZIP transcription factor NRL uncovers the role of c-Jun in early differentiation of rod photoreceptors in the mammalian retina.

The basic motif-leucine zipper (bZIP) transcription factor neural retina leucine zipper (NRL) determines rod photoreceptor cell fate during retinal development, and its loss leads to cone-only retina in mice. NRL works synergistically with homeodomain protein Cone-Rod Homeobox and other regulatory factors to control the transcription of most genes associated with rod morphogenesis and functional maturation, which span over a period of several weeks in the mammalian retina. We predicted that NRL gradually establishes rod cell identity and function by temporal and dynamic regulation of stage-specific transcriptional targets. Therefore, we mapped the genomic occupancy of NRL at four stages of mouse photoreceptor differentiation by CUT&RUN analysis. Dynamics of NRL binding revealed concordance with the corresponding changes in transcriptome of the developing rods. Notably, we identified c-Jun proto-oncogene as one of the targets of NRL, which could bind to specific cis-elements in the c-Jun promoter and modulate its activity in HEK293 cells. Coimmunoprecipitation studies showed the association of NRL with c-Jun, also a bZIP protein, in transfected cells as well as in developing mouse retina. Additionally, shRNA-mediated knockdown of c-Jun in the mouse retina in vivo resulted in altered expression of almost 1000 genes, with reduced expression of phototransduction genes and many direct targets of NRL in rod photoreceptors. We propose that c-Jun-NRL heterodimers prime the NRL-directed transcriptional program in neonatal rod photoreceptors before high NRL expression suppresses c-Jun at later stages. Our study highlights a broader cooperation among cell-type restricted and widely expressed bZIP proteins, such as c-Jun, in specific spatiotemporal contexts during cellular differentiation.

Connexin45 colocalization patterns in the plexiform layers of the developing mouse retina.

Chemical and electrical synapses (gap junctions) are widely prevalent in the nervous system. Gap junctions emerge long before chemical synapses, allowing communication between developing cells, and are thought to be involved in establishing neural circuits. Mounting evidence indicates that these two modalities of synaptic transmission closely interact during retinal development and that such interactions play a critical role in synaptogenesis and circuit formation during the perinatal period. In vertebrates, gap junctions consist of two connexins which in turn are made up of six connexins (Cx). To what extent Cx45 and Cx36, the most abundant connexins in the retina, are involved in synaptogenesis and retinal circuit formation is not known. The here presented immunohistochemical study used stainings of Cx45, Cx36 and Synaptophysin in the outer and inner (IPL) plexiform layers of postnatal day 8-16 mice retinas to shed light on the role of connexins during critical neuronal developmental processes. Cx45 and Cx36 expressions in both plexiform layers of the mouse retina increased till eye opening and dropped afterwards. The percentage of heterotypic Cx45/Cx36 gap junctions is also higher before the critical event of eye opening. Finally, Cx45 is closely located and/or colocalized with Synaptophysin also shortly before eye opening in the IPL of the mouse retina. All findings point towards a pivotal role for Cx45 during postnatal synaptogenesis in the mouse retina. However, a more functional study is needed to determine the role of Cx45 during synaptogenesis and circuit formation.

Gata3 Silencing Is Involved in Neuronal Differentiation and Its Abnormal Expression Impedes Neural Activity in Adult Retinal Neurocytes.

GATA binding protein 3 (Gata3), a zinc-finger transcription factor, plays an important role in neural development. However, its expression and bioactivity in the retina remain unclear. In the present study, our data indicated that Gata3 maintains the precursor state of 661W cells, and Gata3 silencing induces cell differentiation. The expression of Nestin, a marker of precursor cells, was significantly decreased in parallel, whereas the expression of Map2, a marker of differentiated neurons, was significantly increased following the decrease in Gata3. Neurite outgrowth was increased by 2.78-fold in Gata3-silenced cells. Moreover, Gata3 expression generally paralleled that of Nestin in developing mouse retinas. Both Gata3 and Nestin were expressed in the retina at postnatal day 1 and silenced in the adult mouse retina. Exogenous Gata3 significantly inhibited the neural activity of primary retinal neurocytes (postnatal day 1) by decreasing synaptophysin levels, neurite outgrowth, and cell viability. Furthermore, in vivo, exogenous Gata3 significantly induced apoptosis and the contraction of retinal outlay filaments and decreased the a- and b-waves in adult mouse intravitreal injected with AAV-Re-Gata3-T2A-GFP. Thus, Gata3 silencing promotes neuronal differentiation and neurite outgrowth. Its abnormal expression impedes neural activity in adult retinal neurocytes. This study provides new insights into Gata3 bioactivity in retinal neurocytes.

Genome-wide characterization of RNA editing highlights roles of high editing events of glutamatergic synapse during mouse retinal development

Adenosine-to-inosine (A-to-I) RNA editing leads to functional change of neurotransmitter receptor which is essential for neurotransmission and normal neuronal development. As a highly accessible part of central nervous system, retina has been extensively studied, however, it remains largely unknown how RNA editing regulates its development. Here, a genome-wide screening of high-confidence RNA editing events were performed to decipher the dynamic transcriptome regulation by RNA editing during mouse retinal development. 2000 high-confidence editing sites across eight developmental stages of retina were called. Three unique patterns (RNA-editinghigh pattern, RNA-editingmedium pattern and RNA-editinglow pattern) were identified by clustering these editing sites based on their editing level during retinal development. Editing events from RNA-editinghigh pattern were significantly associated with glutamate receptors and regulated synaptic transmission. Interestingly, most non-synonymous high-editing sites were mapped to ion channel genes of glutamatergic synapse which were associated with neurotransmission by controlling ion channel permeability and affecting exocytosis. Meanwhile, these non-synonymous editing sites were evolutionarily conserved and exhibited a consistently increasing editing levels between mouse and human retinal development. Single-cell RNA-seq data analysis revealed that RNA editing events prefer to occur in two main cell types including bipolar and amacrine cells. Genes with non-synonymous high-editing sites were enriched in both bipolar cells and retina ganglion cells, which may mediate retina ganglion cell differentiation by altering channel ion permeability. Together, our results provide novel insights into mechanism of post-transcriptional regulation during retinal development and help to develop novel RNA editing-guided therapeutic strategies for retinal disorders.