Association of SIX1-SIX6 polymorphisms with peripapillary retinal nerve fibre layer thickness in children

PurposeAssociation of SIX1-SIX6 variants with peripapillary retinal nerve fibre layer (p-RNFL) thickness had been reported in adults. This study aimed to investigate these associations in children, with further explorations by spatial, age and sex stratifications.Methods2878 school children aged between 6 and 9 years were enrolled from the Hong Kong Children Eye Study. Three single-nucleotide polymorphisms (SNPs) at the SIX1-SIX6 locus were genotyped. The association of each SNP with p-RNFL thickness (including global and sectoral thickness) were evaluated using multiple linear regression.ResultsSNPs rs33912345 (p=7.7×10−4) and rs10483727 (p=0.0013) showed significant associations with temporal-inferior p-RNFL thickness. The C allele of rs33912345 was associated with a thinner temporal-inferior p-RNFL by an average of 2.44 µm, while rs10483727-T was associated with a thinner temporal-inferior p-RNFL by 2.32 µm. The association with temporal-inferior p-RNFL was the strongest in the 8–9 year-old group for rs33912345 (p=5.2×10−4) and rs10483727 (p=3.3×10−4). Both SNPs were significantly associated with temporal-inferior p-RNFL thickness in boys (p<0.0017), but not in girls (p>0.05). In contrast, rs12436579-C (β=1.66; p=0.0059), but not rs33912345-C (β=1.31; p=0.052) or rs10483727-T (β=1.19; p=0.078), was nominally associated with a thicker nasal-inferior p-RNFL.ConclusionsBoth rs33912345 and rs10483727 at SIX1-SIX6 were associated with p-RNFL thickness in children, especially at the temporal-inferior sector, with age-dependent and sex-specific effects. SNP rs12436579 was associated with nasal-inferior p-RNFL thickness. Our findings suggested a role of SIX1-SIX6 in RNFL variation during neural retina development in childhood.

Wnt1-Cre mediated deletion of BMP7 suggests a role for neural crest-derived BMP7 in retina development and function

Neural crest (NC) contributes to various structures of the eye including cornea, ciliary body and retina. The association of NC-derived cells with hyaloid vessels in the form of pericytes is established. Similarly, persistence of NC-derived cells in the inner retina layer of the mature retina has been suggested. To date, no specific function has been attributed to them. NC-derived Bone morphogenetic protein 7 (BMP7) controls neurogenic properties in the brain and regulates glia differentiation. Here, we assessed the role of NC-derived BMP7 in the adult retina. BMP7 expression was determined using Bmp7LacZ reporter mice. BMP7 was expressed in GCL, IPL, OPL, and photoreceptors in P0, P14 and P30 retinas. Lineage tracing confirmed the presence of NC-derived cells in the GCL, INL, and ONL. Some but not all cells associated with vasculature. To test the function of NC-derived Bmp7, Bmp7fl/flWnt1cre (Bmp7ncko) mice were assessed by histological and functional methods. Loss of NC-derived cells in the GCL and INL and mild structural abnormalities were observed in the Bmp7ncko retina. Electroretinography revealed reduced a wave under photopic conditions and b wave under both scotopic and photopic conditions. The neuronal circuitry in the inner retina appeared affected, evidenced by decreased Calbindin in the GCL, IPL and INL. In the outer retina, S-opsin was increased. BMP7 expression in the mutant retina was strongly decreased at birth, but increased expression from cells other than NC was observed in the adult retina. This was associated with an increase in IBA1, suggestive that loss of NC-derived BMP7 predisposes to development of gliosis-like changes in the adult retina. Overall, our data reveal an important contribution of NC-derived BMP7 for the development and function of the inner and outer retina.

Whole transcriptome sequencing identifies key circRNAs, lncRNAs, and miRNAs regulating neurogenesis in developing mouse retina

Background The molecular complexity of neural retina development remains poorly studied. Knowledge of retinal neurogenesis regulation sheds light on retinal degeneration therapy exploration. Therefore, we integrated the time-series circRNA, lncRNA, miRNA, and mRNA expression profiles of the developing retina through whole-transcriptome sequencing. The key functional ncRNAs and the ceRNA network regulating retinal neurogenesis were identified. Results Transcriptomic analysis identified circRNA as the most variable ncRNA subtype. We screened a series of neurogenesis-related circRNAs, lncRNAs, and miRNAs using different strategies based on their diversified molecular functions. The expression of circCDYL, circATXN1, circDYM, circPRGRIP, lncRNA Meg3, and lncRNA Vax2os was validated by quantitative real-time PCR. These circRNAs and lncRNAs participate in neurotransmitter transport and multicellular organism growth through the intricate circRNA/lncRNA-miRNA-mRNA network. Conclusion Whole-transcriptome sequencing and bioinformatics analysis systematically screened key ncRNAs in retinal neurogenesis. The validated ncRNAs and their circRNA/lncRNA-miRNA-mRNA network involve neurotransmitter transport and multicellular organism growth during retinal development.

Strip1 regulates retinal ganglion cell survival by suppressing Jun-mediated apoptosis to promote retinal neural circuit formation

In the vertebrate retina, an interplay between retinal ganglion cells (RGCs), amacrine and bipolar cells establishes a synaptic layer called the inner plexiform layer (IPL). This circuit conveys signals from photoreceptors to visual centers in the brain. However, the molecular mechanisms involved in its development remain poorly understood. Striatin-interacting protein 1 (Strip1) is a core component of the STRIPAK complex, and it has shown emerging roles in embryonic morphogenesis. Here, we uncover the importance of Strip1 in inner retina development. Using zebrafish, we show that loss of Strip1 causes defects in IPL formation. In strip1 mutants, RGCs undergo dramatic cell death shortly after birth. Amacrine and bipolar cells subsequently invade the degenerating RGC layer, leading to a disorganized IPL. Thus, Strip1 promotes IPL formation through RGC maintenance. Mechanistically, zebrafish Strip1 interacts with its STRIPAK partner, Striatin3, to promote RGC survival by suppressing Jun-mediated apoptosis. In addition to its function in RGC maintenance, Strip1 is required for RGC dendritic patterning, which likely contributes to proper IPL formation. Taken together, we propose that a series of Strip1-mediated regulatory events coordinates inner retinal circuit formation by maintaining RGCs during development, which ensures proper positioning and neurite patterning of inner retinal neurons.

Development and retinal remodeling in the brown anole lizard (Anolis sagrei)

Background. The fovea, a pit in the retina, is believed to be important for high-acuity vision and is a feature found in the eyes of humans and a limited number of vertebrate species that include certain primates, birds, lizards, and fish. At present, model systems currently used for ocular research lack a foveated retina and studies investigating fovea development have largely been limited to histological and molecular studies in primates. As a result, progress towards understanding the mechanisms involved in regulating fovea development in humans is limited and is completely lacking in other, non-primate, vertebrates. To address this knowledge gap, we provide here a detailed histological atlas of retina and fovea development in the bifoveated Anolis sagrei lizard, a novel reptile model for fovea research. We also further test the hypothesis that retinal remodeling, which leads to fovea formation and photoreceptor cell packing, is related to asymmetric changes in eye shape. Results. Anole retina development follows the conventional spatiotemporal patterning observed in most vertebrates, where retina neurogenesis begins within the central retina, progresses throughout the temporal retina, and concludes in the nasal retina. One exception to this general rule is that areas that give rise to the fovea undergo retina differentiation prior to the rest of the retina. We find that retina thickness changes dynamically during periods of ocular elongation and retraction. During periods of ocular elongation, the retina thins, while during retraction it becomes thicker. Ganglion cell layer mounding is also observed in the temporal fovea region just prior to pit formation. Conclusions. Anole retina development parallels that of humans, including the onset and progression of retinal neurogenesis followed by changes in ocular shape and retinal remodeling that leads to pit formation in the retina. We propose that anoles are an excellent model system for fovea development research.

EML1 is essential for retinal photoreceptor localization and light detection

Calcium regulates the response sensitivity, kinetics and adaptation in photoreceptors. In striped bass cones, this calcium feedback includes direct modulation of the transduction cyclic nucleotide-gated (CNG) channels by the calcium-binding protein CNG-modulin. However, the possible role of EML1, the mammalian homolog of CNG-modulin, in modulating phototransduction in mammalian photoreceptors has not been examined. Here, we used mice expressing mutant Eml1 to investigate its role in the development and function of mouse photoreceptors using immunostaining, in-vivo and ex-vivo retinal recordings, and single-cell suction recordings. We found that the mutation of Eml1 causes significant changes in the mouse retinal structure characterized by mislocalization of rods and cones in the inner retina. Consistent with the fraction of mislocalized photoreceptors, rod and cone-driven retina responses were reduced in the mutants. However, the Eml1 mutation had no effect on the dark-adapted responses of rods in the outer nuclear layer. Notably, we observed no changes in the cone sensitivity in the Eml1 mutant animals, either in darkness or during light adaptation, ruling out a role for EML1 in modulating cone CNG channels. Together, our results suggest that EML1 plays an important role in retina development but does not modulate phototransduction in mammalian rods and cones.

Construction and characterization of EGFP reporter plasmid harboring putative human RAX promoter for in vitro monitoring of retinal progenitor cells identity

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

Profile of miR-23 Expression and Possible Role in Regulation of Glutamic Acid Decarboxylase during Postnatal Retinal Development

As neurotransmitter, GABA is fundamental for physiological processes in the developing retina. Its synthesis enzymes are present during retinal development, although the molecular regulatory mechanisms behind the changes in expression are not entirely understood. In this study, we revealed the expression patterns of glutamic acid decarboxylase 67(GAD67) and its coding gene (GAD1) and its potential miRNA-dependent regulation during the first three postnatal weeks in rat retina. To gain insight into the molecular mechanisms, miRNA-sequencing supported by RT-qPCR and in situ hybridization were carried out. GAD1 expression shows an increasing tendency, peaking at P15. From the in silico-predicted GAD1 targeting miRNAs, only miR-23 showed similar expression patterns, which is a known regulator of GAD1 expression. For further investigation, we made an in situ hybridization investigation where both GAD67 and miR-23 also showed lower expression before P7, with the intensity of expression gradually increasing until P21. Horizontal cells at P7, amacrine cells at P15 and P21, and some cells in the ganglion cell layer at several time points were double labelled with miR-23 and GAD67. Our results highlight the complexity of these regulatory networks and the possible role of miR-23 in the regulation of GABA synthesizing enzyme expression during postnatal retina development.

Transcriptome Profiling of Embryonic Retinal Pigment Epithelium Reprogramming

The plasticity of human retinal pigment epithelium (RPE) has been observed during proliferative vitreoretinopathy, a defective repair process during which injured RPE gives rise to fibrosis. In contrast, following injury, the RPE of the embryonic chicken can be reprogrammed to regenerate neural retina in a fibroblast growth factor 2 (FGF2)-dependent manner. To better explore the mechanisms underlying embryonic RPE reprogramming, we used laser capture microdissection to isolate RNA from (1) intact RPE, (2) transiently reprogrammed RPE (t-rRPE) 6 h post-retinectomy, and (3) reprogrammed RPE (rRPE) 6 h post-retinectomy with FGF2 treatment. Using RNA-seq, we observed the acute repression of genes related to cell cycle progression in the injured t-rRPE, as well as up-regulation of genes associated with injury. In contrast, the rRPE was strongly enriched for mitogen-activated protein kinase (MAPK)-responsive genes and retina development factors, confirming that FGF2 and the downstream MAPK cascade are the main drivers of embryonic RPE reprogramming. Clustering and pathway enrichment analysis was used to create an integrated network of the core processes associated with RPE reprogramming, including key terms pertaining to injury response, migration, actin dynamics, and cell cycle progression. Finally, we employed gene set enrichment analysis to suggest a previously uncovered role for epithelial-mesenchymal transition (EMT) machinery in the initiation of embryonic chick RPE reprogramming. The EMT program is accompanied by extensive, coordinated regulation of extracellular matrix (ECM) associated factors, and these observations together suggest an early role for ECM and EMT-like dynamics during reprogramming. Our study provides for the first time an in-depth transcriptomic analysis of embryonic RPE reprogramming and will prove useful in guiding future efforts to understand proliferative disorders of the RPE and to promote retinal regeneration.

Synaptic Remodeling in the Cone Pathway After Early Postnatal Horizontal Cell Ablation

The first synapse of the visual pathway is formed by photoreceptors, horizontal cells and bipolar cells. While ON bipolar cells invaginate into the photoreceptor terminal and form synaptic triads together with invaginating horizontal cell processes, OFF bipolar cells make flat contacts at the base of the terminal. When horizontal cells are ablated during retina development, no invaginating synapses are formed in rod photoreceptors. However, how cone photoreceptors and their synaptic connections with bipolar cells react to this insult, is unclear so far. To answer this question, we specifically ablated horizontal cells from the developing mouse retina. Following ablation around postnatal day 4 (P4)/P5, cones initially exhibited a normal morphology and formed flat contacts with OFF bipolar cells, but only few invaginating contacts with ON bipolar cells. From P15 on, synaptic remodeling became obvious with clustering of cone terminals and mislocalized cone somata in the OPL. Adult cones (P56) finally displayed highly branched axons with numerous terminals which contained ribbons and vesicular glutamate transporters. Furthermore, type 3a, 3b, and 4 OFF bipolar cell dendrites sprouted into the outer nuclear layer and even expressed glutamate receptors at the base of newly formed cone terminals. These results indicate that cones may be able to form new synapses with OFF bipolar cells in adult mice. In contrast, cone terminals lost their invaginating contacts with ON bipolar cells, highlighting the importance of horizontal cells for synapse maintenance. Taken together, our data demonstrate that early postnatal horizontal cell ablation leads to differential remodeling in the cone pathway: whereas synapses between cones and ON bipolar cells were lost, new putative synapses were established between cones and OFF bipolar cells. These results suggest that synapse formation and maintenance are regulated very differently between flat and invaginating contacts at cone terminals.