The histone H3K36 demethylase Fbxl11 plays pivotal roles in the development of retinal late-born cell types.

Histone methylation plays a vital role in retinal development. However, the role of histone H3K36 methylation in retinal development is not clear. We examined the role of H3K36 methylation by loss-of-function analysis of H3K36me1/2 demethylases, Fbxl10, and Fbxl11. We analyzed the effect of knockout of these genes in the developing and mature retina on retinal development. Knockout of Fbxl10 specifically in the developing retina did not result in gross developmental abnormalities. Although adult rod photoreceptor-specific knockout of Fbxl11 in mature retinas did not result in morphological abnormalities, Fbxl11 knockout in developing retinas increased apoptosis, suppressed the proliferation of retinal progenitor cells, and resulted in microphthalmia. Morphological analysis revealed perturbed differentiation of rod photoreceptor and bipolar cells. RNA-seq of retinas at P7 showed markedly decreased expression of genes characterizing rod photoreceptor and bipolar cells in Fbxl11-knockout retinas. In addition, perturbation of alternative splicing increased intron retention in Fbxl11-knockout retinas. Genome-wide evaluation of the H3K36 methylation status revealed that Fbxl11 knockout altered the distribution of H3K36me2/3 in genes important for rod photoreceptor development. Taken together, we show that Fbxl11 plays pivotal roles in the development of retinal late-born cell types and may contribute to tight control of H3K36 methylation during retinal development.

The mechanism of multistep progression of the transcriptional cascade in activated microglia as approached by a proteome approach.

The ocular cytokine network plays pivotal roles in terms of the initiation and progression of retinal degeneration. Several types of immunocompetent cells such as microglia participate in inflammation, and a temporal transition in the molecular events of inflammation has been hypothesized. We previously found that the Csf2 gene was induced in the early phase of retinal degeneration. CSF2 participates in the transcriptional activation of several cytokines expressed by microglia; however, whether CSF2 is essential in this context is not known. In this work, we approach this question by using anti-CSF2 neutralizing bntibody and the protein synthesis inhibitor cycloheximide (CHX). We first revealed that CSF2 positively regulated the cytokine induction cascade using a CSF2-neutralizing antibody (anti-CSF2) to treat the microglial cell line that were activated by lipopolysaccharide (LPS). LPS or Lipid A stimulation in the presence of the protein synthesis inhibitor cycloheximide (CHX) led to cytokine superinduction, but suppression of the expression of a few cytokines was also noted in MG5 cells. To examine transitions of the molecular events within LPS-activated microglia, we next performed proteome analysis of MG5 cells stimulated with LPS for 0, 4, and 9 h. The Database for Annotation, Visualization, and Integrated Discovery analysis of differentially expressed proteins showed that various mRNA-modifying molecules were induced after LPS stimulation, in addition to molecules involved in inflammation. However, the numbers of common proteins founded in the comparison between the induced proteins of 4 and 9 h were only one-third and one-half of induced proteins at 4 and 9 h, respectively, suggesting dynamic transition of the induced proteins. LPS-induced mRNA-modifying proteins were almost completely suppressed by CHX, as expected, suggesting that transient induction of transcription-editing proteins plays an important role in terms of the phenotype of inflammation that develops in microglia after LPS stimulation.

Evaluation of CRISPR/Cas9 exon-skipping vector for choroideremia using human induced pluripotent stem cell-derived RPE.

BACKGROUND: Exon-skipping is a powerful genetic tool, especially when delivering genes using an AAV-mediated full-length gene supplementation strategy is difficult owing to large length of genes. Here, we used engineered human induced pluripotent stem cells and artificial intelligence to evaluate clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9-based exon-skipping vectors targeting genes of the retinal pigment epithelium (RPE). The model system was choroideremia; this is an X-linked inherited retinal disease caused by mutation of the CHM gene. METHODS: We explored whether artificial intelligence detected differentiation of human OTX2, PAX6 and MITF (hOPM) cells, in which OTX2, PAX6 and MITF expression was induced by doxycycline treatment, into RPE. Plasmid encoding CHM exon-skipping modules targeting the splice donor sites of exons 6 were constructed. A clonal hOPM cell line with a frameshift mutation in exon 6 was generated and differentiated into RPE. CHM exon 6-skipping was induced, and the effects of skipping on phagocytic activity, cell death and prenylation of Rab small GTPase (RAB) were evaluated using flow cytometry, an in vitro prenylation assay and western blotting. RESULTS: Artificial intelligence-based evaluation of RPE differentiation was successful. Retinal pigment epithelium cells with a frameshift mutation in exon 6 showed increased cell death, reduced phagocytic activity and increased cytosolic unprenylated RABs only when oxidative stress was in play. The latter two phenotypes were partially rescued by exon 6-skipping of CHM. CONCLUSIONS: CHM exon 6-skipping contributed to RPE phagocytosis probably by increasing RAB38 prenylation under oxidative stress.

Downregulation of VEGF in the retinal pigment epithelium followed by choriocapillaris atrophy after NaIO3 treatment in mice.

Sodium iodate (NaIO3) induces retinal pigment epithelium (RPE) dysfunction, which leads to photoreceptor degeneration. Previously, we used electron microscopy to show that the administration of NaIO3 resulted in the accumulation of cell debris in the subretinal space, which was thought to be caused by failed phagocytosis in the outer segment of the photoreceptor due to RPE dysfunction. We further analyzed the pathological changes in the retina and choroid of NaIO3-injected mice, and found that the expression of OTX2, an RPE marker, disappeared from central part of the RPE 1 day after NaIO3 administration. Furthermore, fenestrated capillaries (choriocapillaris, CC) adjacent to the RPE could not be identified only 2 days after NaIO3 administration. An examination of the expression of the CC-specific protein plasmalemma vesicle-associated protein (PLVAP), in sections and flat-mount retina/choroid specimens showed destruction of the CC, and complete disappearance of the PLVAP signal 7 days after NaIO3 administration. In contrast, CD31 flat-mount immunohistochemistry of the retina indicated no difference in retinal vessels between NaIO3-treated mice and controls. Electron microscopy showed that the fenestrated capillaries in the kidney and duodenum were morphologically indistinguishable between control and NaIO3-treated mice. We examined cytokine production in the retina and RPE, and found that the Vegfa transcript level in the RPE decreased starting 1 day after NaIO3 administration. Taken together, these observations show that NaIO3 reduces the CC in the early stages of the pathology, which is accompanied by a rapid decrease in Vegfa expression in the RPE.

Mapping membrane lipids in the developing and adult mouse retina under physiological and pathological conditions using mass spectrometry.

Membrane phospholipids play pivotal roles in various cellular processes, and their levels are tightly regulated. In the retina, phospholipids had been scrutinized because of their distinct composition and requirement in visual transduction. However, how lipid composition changes during retinal development remains unclear. Here, we used liquid chromatography-mass spectrometry (LC-MS) to assess the dynamic changes in the levels of two main glycerophospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), in the developing mouse retina under physiological and pathological conditions. The total levels of PC and PE increased during retinal development, and individual lipid species exhibited distinct level changes. The amount of very-long-chain PC and PE increased dramatically in the late stages of retinal development. The mRNA levels of Elovl2 and Elovl4, genes encoding enzymes essential for the synthesis of very-long-chain polyunsaturated fatty acids, increased in developing photoreceptors. Cell sorting based on CD73 expression followed by LC-MS revealed distinct changes in PC and PE levels in CD73-positive rod photoreceptors and CD73-negative retinal cells. Finally, using the NaIO3-induced photoreceptor degeneration model, we identified photoreceptor-specific changes in PC and PE levels from 1 day after NaIO3 administration, before the outer segment of photoreceptors displayed morphological impairment. In conclusion, our findings provide insight into the dynamic changes in PC and PE levels in the developing and adult mouse retina under physiological and pathological conditions. Furthermore, we provide evidence that cell sorting followed by LC-MS is a promising approach for investigating the relevance of lipid homeostasis in the function of different retinal cell types.

Roles of CSF2 as a modulator of inflammation during retinal degeneration.

Colony-stimulating factor 2 (CSF2) is a potent cytokine that stimulates myeloid cells, such as dendritic cells and macrophages. We have been analyzing the roles of microglia in retinal degeneration through the modulation of inflammation in the eye, and examined the roles of CSF2 in this process. Both subunits of the CSF2 receptor are expressed in microglia, but no evidence suggesting the involvement of CSF2 in inflammation in the degenerating eye has been reported. We found that Csf2 transcripts were induced in the early phase of in vitro mouse adult retina culture, used as degeneration models, suggesting that CSF2 induction is one of the earliest events occurring in the pathology of retinal degeneration. The administration of CSF2 into the retina after systemic NaIO3 treatment increased the number of microglia. To examine the roles of CSF2 in retinal inflammation, we overexpressed CSF2 in retinal explants. Induction of CSF2 activated microglia and Müller glia, and the layer structure of the retina was severely perturbed. CC motif chemokine ligand 2 (Ccl2) and C-X-C motif chemokine ligand 10 (Cxcl10), both of which are expressed in activated microglia, were strongly induced by the expression of CSF2 in the retina. The addition of CSF2 to primary retinal microglia and the microglial cell lines MG5 and BV2 showed statistically significant increase in Ccl2 and Il1b transcripts. Furthermore, CSF2 induced proliferation, migration, and phagocytosis in MG5 and/or BV2. The effects of CSF2 on microglia were mild, suggesting that CSF2 induced strong inflammation in the context of the retinal environment.

Characterization of human-induced pluripotent stem cells carrying homozygous RB1 gene deletion.

Retinoblastoma is an infant cancer that results from loss of RB1 expression in both alleles. The RB1 gene was the first reported cancer suppressor gene; however, the mechanism by which RB1 loss causes cancer in the retina has not yet been clarified. Human-induced pluripotent stem cells (iPSCs) provide an ideal tool for mechanistic research regarding retinoblastoma. However, because RB1 is a tumor suppressor, loss of both alleles of RB1 in human iPS cells may affect the phenotype of the cells. To examine this possibility, we established human iPSCs with deletions in both alleles of RB1 by CRISPR/Cas9 technique to characterize the associated phenotype. We first examined the expression of RB1 transcripts by RT-qPCR, and RB1 transcripts were expressed in immature hiPSCs and then the expression levels of RB1 transcripts consistently increased during retinal organoid differentiation in human iPSCs. Expression levels of immature markers including SSEA4, OCT3/4 and NANOG were indistinguishable between control iPSCs and RB1 knockout iPSCs. Proliferative activity was also unaffected by homozygous RB1 deletion. Taken together, we showed that homozygous deletion of RB1 did not affect the maturation and proliferation statuses of human iPSCs.

H3K27me3 demethylase UTX regulates the differentiation of a subset of bipolar cells in the mouse retina.

Di- and trimethylation of lysine 27 on histone 3 (H3K27me2/3) is a critical gene repression mechanism. We previously showed that down-regulation of the H3K27 demethylase, Jumonji domain-containing protein 3 (JMJD3), resulted in a reduced number of protein kinase C (PKC)α-positive rod ON-bipolar cells. In this work, we focused on the role of another H3K27 demethylase, ubiquitously transcribed tetratricopeptide repeat X chromosome (UTX), in retinal development. UTX was expressed in the retinal progenitor cells of the embryonic mouse retina and was observed in the inner nuclear layer during late retinal development and in the mature retina. The short hairpin RNA-mediated knockdown of Utx in a mouse retinal explant led to a reduced number of PKCα-positive rod ON-bipolar cells. However, other retinal subtypes were unaffected by this knockdown. Using a retina-specific knockout of Utx in mice, the in vivo effects of UTX down-regulation were examined. Again, the number of PKCα-positive rod ON-bipolar cells was reduced, and no other apparent phenotypes, including retinal progenitor proliferation, apoptosis or differentiation, were observed. Finally, we examined retina-specific Utx and Jmjd3 double-knockout mice and found that although the number of rod ON-bipolar cells was reduced, no additional effects from the loss of Utx and Jmjd3 were observed. Taken together, our data show that UTX contributes to retinal differentiation in a lineage-specific manner.

Pivotal roles of Fezf2 in differentiation of cone OFF bipolar cells and functional maturation of cone ON bipolar cells in retina.

During development of the retina, common retinal progenitor cells give rise to six classes of neurons that subsequently further diversify into more than 55 subtypes of neuronal subtypes. Here, we have investigated the expression and function of Fezf2, Fez zinc finger family of protein, in the developing mouse retina. Expression of Fezf2 transcripts was strongly observed in the embryonic retinal progenitors at E14.5 and declined quickly in subsequent development of retina. Then, in postnatal stage at around day 8, Fezf2 was transiently expressed then declined again. Loss-of-function analysis using retinas from mice in which Fezf2 coding region was substituted with ß-galactosidase showed that Fezf2 is expressed in a subset of cone OFF bipolar cells and required for their differentiation. Using electroretinogram, we found that Fezf2 knockout retina exhibited significantly reduced photopic b-wave, suggesting functional abnormality of cone ON bipolar cells. Furthermore, reduced expression of synaptic protein Trpm1 and structural alteration of ON bipolar cell invagination, both of which affected cone photoreceptor terminal synaptic activity, was identified by transmission electron microscopy and immunohistochemistry, respectively. Taken together, our results show that Fezf2 is indispensable in differentiation of bipolar precursors into cone OFF bipolar cells and in functional maturation of cone ON bipolar cells during development of mouse retina. These results contribute to our understanding of how diversity of neuronal subtypes and hence specificity of neuronal connections are established in the retina by intrinsic cues.

Histone demethylase Jmjd3 is required for the development of subsets of retinal bipolar cells.

Di- and trimethylation of lysine 27 on histone H3 (H3K27me2/3) is an important gene repression mechanism. H3K27me2/3-specific demethylase, Jmjd3, was expressed in the inner nuclear layer during late retinal development. In contrast, H3K27 methyltransferase, Ezh2, was highly expressed in the embryonic retina but its expression decreased rapidly after birth. Jmjd3 loss of function in the developing retina resulted in failed differentiation of PKC-positive bipolar cell subsets (rod-ON-BP) and reduced transcription factor Bhlhb4 expression, which is critical for the differentiation of rod-ON-BP cells. Overexpression of Bhlhb4, but not of other BP cell-related genes, such as transcription factors Neurod and Chx10, in Jmjd3-knockdown retina rescued loss of PKC-positive BP cells. Populations of other retinal cell subsets were not significantly affected. In addition, proliferation activity and apoptotic cell number during retinal development were not affected by the loss of Jmjd3. Levels of histone H3 trimethyl Lys27 (H3K27me3) in the Bhlhb4 locus were lower in Islet-1-positive BP cells and amacrine cells than in the Islet-1-negative cell fraction. The Islet-1-negative cell fraction consisted mainly of photoreceptors, suggestive of lineage-specific demethylation of H3K27me3 in the Bhlhb4 locus. We propose that lineage-specific H3K27me3 demethylation of critical gene loci by spatiotemporal-specific Jmjd3 expression is required for appropriate maturation of retinal cells.

Ablation of Kcnj10 expression in retinal explants revealed pivotal roles for Kcnj10 in the proliferation and development of Müller glia.

PURPOSE: We previously found that Kcnj10, an inwardly-rectifying potassium channel, is a gene expressed in c-kit-positive retinal progenitor cells on P1. The shRNA-mediated screening of the functions of the genes for retinal development in retinal explant culture suggested a role for Kcnj10 in the differentiation of 23Müller glia. In the present study, we extended the work and focused on analyzing the role of Kcnj10 in retinal development. METHODS: shRNA-mediated downregulation of Kcnj10 in retinal explants and the in vivo mouse retina at the P1 stage was performed. Differentiation and proliferation of the retina were examined with immunohistochemistry. The effect of barium (Ba(2+)) treatment, which inhibits potassium currents by blocking potassium channels, on retinal development was examined. RESULTS: When Kcnj10 was downregulated at E18, cellular proliferation and morphological differentiation were perturbed; in particular, a decreased number of Müller glial cells with abnormal morphological maturation was observed. The overexpression of Kcnj10 in retinal progenitors did not result in gross abnormality during retinal development, but rescued the abnormal differentiation induced with sh-Kcnj10. The presence of Ba(2+) in the retinal explant medium led to a phenotype similar to that seen with sh-Kcnj10. Ba(2+) exerts an effect mainly during late retinal development, and sh-Kcnj10 in the P1 retina affected Müller glia maturation, suggesting that Kcnj10 plays a pivotal role in the maturation of retinal cell subsets. A previous study of Kcnj10-knockout mice showed no obvious abnormality in retinal differentiation, especially of Müller glia. We examined the effects of the downregulation of Kcnj10 with in vivo electroporation of sh-Kcnj10 in the P1 retina. Retinal differentiation was perturbed, as seen following the in vitro downregulation of Kcnj10, suggesting that compensatory gene expression and/or signaling occurred in the Kcnj10-knockout mice in the retina, leading to normal eye development. CONCLUSION: Kcnj10 plays a role in Müller glia maturation during retinal development probably through ionic channel activities.

The role of lipid peroxidation in epithelial-mesenchymal transition of retinal pigment epithelial cells.

Epithelial-Mesenchymal Transition (EMT) of retinal pigment epithelial (RPE) cells is recognized as pivotal in various retinal diseases. Previous studies have suggested a reciprocal regulation between reactive oxygen species (ROS) and EMT, though the involvement of peroxidized lipids or the effects of reducing them has remained unclear. The present study disclosed that EMT of ARPE-19 cells induced by TGF-ß2 and TNF-α involves increased lipid peroxidation, and Ferrostatin-1 (Fer-1), a lipophilic antioxidative agent, successfully inhibited the increase in lipid peroxidation. Fer-1 suppressed the formation of EMT-associated fibrotic deposits, while EMT induction or Fer-1 treatment did not influence the cell viability or proliferation. Functionally, Fer-1 impeded EMT-driven cell migration and reduction in transepithelial electrical resistance. It demonstrated regulatory prowess by downregulating the mesenchymal marker fibronectin, upregulating the epithelial marker ZO-1, and inhibiting the EMT-associated transcriptional factor ZEB1. Additionally, VEGF, a major pathogenic cytokine in various retinal diseases, is also upregulated during EMT, and Fer-1 significantly mitigated the effect. The present study disclosed the involvement of lipid peroxidation in EMT of RPE cells, and suggests the suppression of lipid peroxidation may be a potential therapeutic target in retinal diseases in which EMT is implicated.

Setd5, but not Setd2, is indispensable for retinal cell survival and proliferation.

Trimethylation of histone H3 at lysine 36 (H3K36me3) is associated with active transcription. We used mouse retinal explant cultures and shRNA to investigate the roles of Setd2 and Setd5, which encode H3K36me3 methyltransferases, in retinal development. We found that shSetd5 caused abnormal retinal structures and reduced rods and Müller cells, whereas shSetd2 did not cause any abnormalities. The mutant SETD5 lacking the SET domain failed to reverse the phenotypes observed in the shSetd5-expressing retinas, while SETD5S1257*, which does not interact with HDAC3 and PAF1 complexes, rescued proliferation, but not apoptosis, induced by shSetd5. Taken together, we found that Setd5, but not Setd2, is essential for sustaining retinal cell survival and proliferation, and the SET domain of SETD5 is pivotal for both functions.

Setd1a Plays Pivotal Roles for the Survival and Proliferation of Retinal Progenitors via Histone Modifications of Uhrf1.

Purpose: The trimethylation of histone H3 at lysine 4 (H3K4me3) facilitates transcriptional gene activation, and Setd1a is the methyltransferase specific to H3K4. H3K4me3 has been reported to regulate rod photoreceptor differentiation; however, the roles H3K4me3 plays in retinal progenitor cell (RPC) proliferation and differentiation during early retinal development remain unclear. Methods: Using an in vitro retinal explant culture system, we suppressed the expression of Setd1a by introducing shSetd1a. We examined the expression level and H3K4me3 level of genes by RNA Sequencing and ChIP assay, respectively. Results: We found that Setd1a depletion resulted in increased apoptosis and proliferation failure in late RPCs. Expression of wild-type SETD1A, but not SETD1A that lacked the catalytic SET domain, reversed the shSetd1a-induced phenotype. RNA Sequencing revealed that proliferation-related genes were downregulated upon shSetd1a expression. Based on publicly available H3K4me3-ChIP sequencing data of retinal development, we identified Uhrf1 as a candidate target gene of Setd1a. The expression of shSetd1a led to a decrease in Uhrf1 transcript levels and reduced H3K4me3 levels at the Uhrf1 locus. Increased apoptosis and the suppression of proliferation in late RPCs were observed in retinal explants expressing shUhrf1, similar to the outcomes observed in shSetd1a-expressing retinas. The overexpression of UHRF1 did not rescue shSetd1a-induced apoptosis, but reversed the suppression of proliferation. Conclusions: These results indicate that Setd1a contributes to the survival and proliferation of retinal cells by regulating histone methylation, Setd1a regulates Uhrf1 expression, and these two molecules cooperate to regulate RPC survival and proliferation.

Jmjd3 Plays Pivotal Roles in the Proper Development of Early-Born Retinal Lineages: Amacrine, Horizontal, and Retinal Ganglion Cells.

Purpose: Trimethylation of histone H3 at lysine 27 (H3K27me3) is a critical mediator of transcriptional gene repression, and Jmjd3 and Utx are the demethylases specific to H3K27me3. Using an in vitro retinal explant culture system, we previously revealed the role of Jmjd3 in the development of rod bipolar cells; however, the roles of Jmjd3 in the development of early-born retinal cells are unknown due to limitations concerning the use of retinal explant culture systems. In this study, we investigated the roles of Jmjd3 in the development of early-born retinal cells. Methods: We examined retina-specific conditional Jmjd3 knockout (Jmjd3-cKO) mice using immunohistochemistry and quantitative reverse transcription PCR and JMJD3 binding to a target locus by chromatin immunoprecipitation analysis. Results: We observed reductions in amacrine cells (ACs) and horizontal cells (HCs), as well as lowered expression levels of several transcription factors involved in the development of ACs and HCs in the Jmjd3-cKO mouse retina. JMJD3 bound the promoter regions of these transcription factors. Notably, an elevated number of retinal ganglion cells (RGCs) was observed at embryonic stages, whereas RGCs were moderately reduced at later postnatal stages in the Jmjd3-cKO retina. We also observed reduced expression of Eomes, which is required for the maintenance of RGCs, as well as lower H3K27me3 level and lower JMJD3 binding in the promoter region of Eomes in RGC-enriched cells. Conclusions: The results indicated that Jmjd3 has critical roles in the development of early-born retinal subtypes, and suggested biphasic roles of Jmjd3 in RGC production and maintenance.

RasV12 Expression in Microglia Initiates Retinal Inflammation and Induces Photoreceptor Degeneration.

Purpose: The role of activated retinal microglia in driving retinal degeneration has been implicated in a number of in vivo disease models. Here, we investigated the primary consequences of microglial activation by the specific expression of constitutively active Ras in microglia in a transgenic mouse model before the onset of any degenerative changes in the retina. Methods: The double transgenic lines CAG-LSL-RasV12-IRES-EGFP; Cx3cr1CreER/+ (Cx3cr1-RasV12 mice) and CAG-LSL-EGFP; Cx3cr1CreER_+ (control mice) were generated. The expression of RasV12 was induced in microglia by tamoxifen administration, and the retinas were examined by immunohistochemistry of frozen sections, RT-qPCR, and live imaging. Results: RasV12 expression in retinal microglial cells promoted cell proliferation, cytokine expression, and phagocytosis. RasV12-expressing microglia migrated toward the inner and outer layers of the retina. Examination of glial fibrillary acidic protein (GFAP) expression revealed activation of Müller glia in the retina. We also observed loss of the photoreceptors in the outer nuclear layer in close proximity to microglial cells. However, no significant neurodegeneration was detected in the inner nuclear layer (INL) or ganglion cell layer (GCL). The morphology of RasV12-expressing microglia in the GCL and INL retained more ramified features compared with the predominantly-ameboid morphology found in outer retinal microglia. Conclusions: The expression of RasV12 is sufficient to activate microglia and lead to photoreceptor degeneration. Neurons in the inner side of the retina were not damaged by the RasV12-activated microglia, suggesting that microenvironment cues may modulate the microglial phenotypic features and effects of microglial activation.

Cd9 Protects Photoreceptors from Injury and Potentiates Edn2 Expression.

Purpose: Cd9 is a tetraspanin membrane protein that plays various roles in tissue development and disease pathogenesis, especially in cancer, but the expression patterns and function of Cd9 in retinal development and disease are not well understood. We asked its roles during retinal photoreceptor degeneration by using CD9-knockout mice. Methods: Cd9 knockout mice and rd1 mice were used to examine roles of Cd9 for progression of photoreceptor degeneration. Reverse transcription-polymerase chain reaction and immunohistochemistry were mainly used as analytical methods. Results: Cd9 transcripts were only weakly expressed in retina at embryonic day 14, but its expression level subsequently increased and peaked at around postnatal day 12. In 6-week-old female mice derived retina, mRNA expression decreased slightly but was maintained at a significant level. Published RNA-sequencing data and immunohistochemistry indicated that Cd9 was expressed abundantly in Müller glia and weakly in other retinal neurons. Notably, when photoreceptors were damaged, Cd9 expression was increased in rod photoreceptors and decreased in Müller glia. Cd9 knockout mice retinas developed normally; however, once the retina suffered damage, degeneration of photoreceptors was more severe in Cd9 knockout retinas than control retinas. Induction of Edn2, which is known to protect against photoreceptor damage, was severely hampered. In addition, induction of Socs3, which is downstream of gp130 (Il6st), was weaker in Cd9 knockout retinas. Conclusions: Taken together, these findings indicate that, although Cd9 was dispensable for normal gross morphological development, it protected rod photoreceptors and enhanced Edn2 expression, possibly through modulation of gp130 signaling.

Molecular mechanisms of H3K27me3 and H3K4me3 in retinal development.

The retina consists of six types of neuron and Müller glia, and they are individually derived from common retinal progenitors in a chronologically defined order. Therefore, the signaling environment and competency of retinal progenitors change during retinal development, and the retina serves as an excellent model system to analyze molecular events during development. Much attention has been given to the identification of transcription factors and epigenetic mechanisms. The dynamic changing of the histone modification levels of retina-specific genes has been observed, and the modification patterns of H3K4me3 and H3K27me3 are regulated in a retinal cell type-specific manner. Therefore, it appears that the dynamism of histone modification in the developing retina is regulated both chronologically and in a cell type-specific manner in a particular gene category. Loss- and gain-of-function analyses of enzymes involved in the methylation and demethylation of H3K4 and K27 in the retina have indicated their critical roles in proliferation, differentiation, and determinations of the timing for differentiation. We summarize recent findings related to the roles of H3K4me3 and H3K27me3 in retinal development to discuss how the retinal system provides intriguing data on and contributes to concepts regarding the roles of histone modification in the chronological regulation of tissue development.

Roles of Nmnat1 in the survival of retinal progenitors through the regulation of pro-apoptotic gene expression via histone acetylation.

Leber congenital amaurosis (LCA) is a severe, genetically heterogeneous dystrophy of the retina and mutations in the nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) gene is one of causal factors of LCA. NMNAT1 is a nuclear enzyme essential for nicotinamide adenine dinucleotide (NAD) biosynthesis pathways, but the mechanisms underlying the LCA pathology and whether NMNAT1 has a role in normal retinal development remain unclear. Thus, we examined the roles of Nmnat1 in retinal development via short hairpin (sh)-RNA-mediated downregulation. Retinal explants expressing sh-Nmnat1 showed large numbers of apoptotic retinal progenitor cells in the inner half of the neuroblastic layer. Decreased intracellular NAD content was observed and the addition of NAD to the culture medium attenuated sh-Nmnat1-induced apoptosis. Of the nuclear Sirtuin (Sirt) family, the expression of sh-Sirt1 and sh-Sirt6 resulted in a phenotype similar to that of sh-Nmnat1. Sirt proteins are histone deacetylases and the expression of sh-Nmnat1 increased the levels of acetylated histones H3 and H4 in the retina. Expression of sh-Nmnat1 resulted in significantly increased expression of Noxa and Fas, two pro-apoptotic genes. Acetylation of the genomic 5'-untranslated regions of Noxa and Fas loci was upregulated by sh-Nmnat1 expression. The co-expression of sh-Fas with sh-Nmnat1 reduced the number of apoptotic cells induced by sh-Nmnat1 expression alone. Taken together, our data suggested that the increased expression of Noxa and Fas explains, at least in part, the phenotype associated with sh-Nmnat1 in the retina. Taken together, these findings demonstrate the importance of the NAD biosynthesis pathway in normal development of the retina.