Yukihiro Baba

Dicer plays essential roles for retinal development by regulation of survival and differentiation.

PURPOSE Much attention has been paid to the roles of microRNA in developmental and biological processes. Dicer plays essential roles in cell survival and proliferation in various organs. We examined the role of Dicer in retinal development using retina-specific conditional knockout of Dicer in mice. METHODS Dkk3-Cre expressed the Cre gene in retinal progenitor cells from an early embryonic stage. The authors analyzed Dkk-Cre/Dicer-flox (Dicer-CKO) mice for their survival, proliferation, and differentiation. To analyze the role of Dicer in later stages of retinal development, a Cre expression plasmid was introduced into the neonatal retina by electroporation, and retinal differentiation was examined. RESULTS Dicer-CKO mice were born at the numbers we expected, based on Mendelian genetics, but their eyes never opened. Massive death of retinal progenitor cells occurred during embryogenesis, resulting in microphthalmia, and most retinal cells had disappeared by postnatal day 14. In vitro reaggregation culture of Dicer-CKO retinal cells showed that cell death and the suppression of proliferation by Dicer inactivation occurred in a cell-autonomous manner. Cell differentiation markers were expressed in the Dicer-CKO retina; however, these cells localized abnormally, and the inner plexiform layer was absent, suggesting that cell migration and morphologic differentiation, especially process extension, were perturbed. Forced neonatal expression of Cre induced apoptosis and affected the expression of differentiation markers. CONCLUSIONS Taken together, these results show that Dicer is essential during early retinal development.

Roles of histone H3K27 trimethylase Ezh2 in retinal proliferation and differentiation.

The histone modification H3K27me3 regulates transcription negatively, and Jmjd3 and Ezh2 demethylate and methylate H3K27me3 and H3K27, respectively. We demonstrated previously that Jmjd3 plays pivotal roles in the differentiation of subsets of bipolar (BP) cells by regulating H3K27me3 levels at the Bhlhb4 and Vsx1 loci, both of which are transcription factors essential for the maturation of BP cell subsets. In this study, we examined the role of Ezh2 in retinal development using retina‐specific Ezh2 conditional knockout mice (Ezh2‐CKO). The eyes of the Ezh2‐CKO mice were microphthalemic, and the proliferation of retinal cells was diminished postnatally in Ezh2‐CKO. Differentiation of all examined retinal subsets was observed with higher proportion of BP cell subsets, which was determined by immunostaining using specific retinal markers. The onsets of Müller glia and rod photoreceptor differentiation were accelerated. The expression of Bhlhb4 was increased in postnatal retinas, which was accompanied by the loss of H3K27me3 modifications at these genetic loci. Decreased expression of proneural genes in postnatal stage was observed. As reported previously in other Ezh2‐KO tissues, increased expression of Arf/Ink4a was observed in the Ezh2‐CKO retinas. The ectopic expression of Arf or Ink4a in the retina suppressed proliferation and increased apoptosis. In addition, earlier onset of Müller glia differentiation was observed in Ink4a‐expressing cells. These results support an important role for histone H3K27me3 modification in regulating the proliferation and maturation of certain subsets of interneurons in the retina.

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

Significance The role of repressive gene regulation through histone modifications is known in various biological processes. Histone H3 trimethyl Lys27 (H3K27me3) represses gene expression, and our goal was to understand how this methylation regulates cell differentiation in the vertebrate retina. In this work, we focused on the role of demethylase (Jmjd3) of H3K27me3 in retinal development. Spatiotemporal expression patterns of Jmjd3 during retinal development were shown. Suppression of Jmjd3 expression during retinal development resulted in the failure of differentiation of retinal cell subsets. Lowered expression of genes essential for differentiation of the subsets by loss of expression of Jmjd3 was observed. Therefore, we propose that lineage-specific H3K27me3 demethylation of critical gene loci by spatiotemporal-specific Jmjd3 expression is required for appropriate maturation of retinal 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.

Conditional rod photoreceptor ablation reveals Sall1 as a microglial marker and regulator of microglial morphology in the retina.

Neurodegeneration has been shown to induce microglial activation and the infiltration of monocyte‐derived macrophages into the CNS, resulting in the coexistence of these two populations within the same lesion, though their distinct features remain elusive. To investigate the impact of rod photoreceptor degeneration on microglial activation, we generated a toxin‐mediated genetic model of rod degeneration. Rod injury induced microglial proliferation and migration toward the photoreceptors. Bone marrow transplantation revealed the invasion of monocyte‐derived macrophages into the retina, with microglia and the infiltrating macrophages showing distinct distribution patterns in the retina. By comparing the gene expression profiles of the activated microglia and infiltrating macrophages, we identified microglia‐specific genes, including Ak1, Ctsf, Sall1, Phlda3, and Spns2. An analysis of Sall1gfp knock‐in mice showed GFP expression in the microglia of developing and mature healthy retinas. DTA injury induced the expansion of Sall1gfp+ microglia, whereas Ly6C+ monocyte‐derived macrophages were mostly Sall1gfp‐, supporting the idea that Sall1 is exclusively expressed in microglia within the retinal phagocyte pool. We evaluated the contribution of microglia to the phagocyte pool in rd1 mutant retinas and found that Sall1gfp+ microglia constituted the majority of phagocytes. A Sall1 deficiency did not affect microglial colonization of the retina and the cortex, but it did change their morphology from a ramified to a more amoeboid appearance. The morphological defects observed in Sall1‐deficient microglia were not rescued by the presence of wild‐type non‐microglial cells, suggesting that Sall1 functions cell‐autonomously in microglia. Taken together, our data indicate that Sall1 regulates microglial morphology during development. GLIA 2016;64:2005–2024

The role of Zic family zinc finger transcription factors in the proliferation and differentiation of retinal progenitor cells

Members of the Zic family of zinc finger transcription factors play critical roles in a variety of developmental processes. Using DNA microarray analysis, we found that Zics are strongly expressed in SSEA-1-positive early retinal progenitors in the peripheral region of the mouse retina. Reverse-transcription polymerase chain reaction using mRNA from the retina at various developmental stages showed that Zic1 and Zic2 are expressed in the embryonic retina and then gradually disappear during retinal development. Zic3 is also expressed in the embryonic retina; its expression level slightly decreases but it is expressed until adulthood. We overexpressed Zic1, Zic2, or Zic3 in retinal progenitors at embryonic day 17.5 and cultured the retina as explants for 2 weeks. The number of rod photoreceptors was fewer than in the control, but no other cell types showed significant differences between control and Zic overexpressing cells. The proliferation activity of normal retinal progenitors decreased after 5 days in culture, as observed in normal in vivo developmental processes. However, Zic expressing retinal cells continued to proliferate at days 5 and 7, suggesting that Zics sustain the proliferation activities of retinal progenitor cells. Since the effects of Zic1, 2, and 3 are indistinguishable in terms of differentiation and proliferation of retinal progenitors, the redundant function of Zics in retinal development is suggested.

Transition of differential histone H3 methylation in photoreceptors and other retinal cells during retinal differentiation

To analyze cell lineage-specific transitions in global transcriptional and epigenetic changes during retinogenesis, we purified retinal cells from normal mice during postnatal development into two fractions, namely, photoreceptors and other retinal cells, based on Cd73 expression, and performed RNA sequencing and ChIP sequencing of H3K27me3 and H3K4me3. Genes expressed in the photoreceptor lineage were marked with H3K4me3 in the Cd73-positive cell fraction; however, the level of H3K27me3 was very low in both Cd73-positive and -negative populations. H3K27me3 may be involved in spatio-temporal onset of a subset of bipolar-related genes. Subsets of genes expressed in amacrine and retinal ganglion cells, which are early-born retinal cell types, were suggested to be maintained in a silent state by H3K27me3 during late-stage retinogenesis. In the outer nuclear layer, upregulation of Rho and rod-related genes were observed in Ezh2-ablated retina, suggesting a role for H3K27me3 in the maintenance of proper expression levels. Taken together, our data on the transition of lineage-specific molecular signatures during development suggest that histone methylation is involved in retinal differentiation and maintenance through cell lineage-specific mechanisms.

CD138/syndecan-1 and SSEA-1 mark distinct populations of developing ciliary epithelium that are regulated differentially by Wnt signal.

Ciliary epithelium (CE), which consists of nonpigmented and pigmented layers, develops from the optic vesicle. However, the molecular mechanisms underlying CE development have not been closely examined, in part because cell‐surface markers suitable for specific labeling of subregions of the retina were unknown. Here, we identified CD138/syndecan‐1 and stage specific embryonic antigen‐1 (SSEA‐1) CD15 as cell‐surface antigens marking nonpigmented and pigmented CE, respectively. During retinal development, both CD138 and SSEA‐1 were expressed in the early stage, and segregation of these markers in the tissue began at around embryonic day (E) 10. As a result, CD138‐positive (CD138+) cells were found at the most distal tip of the retina, and SSEA‐1+ cells were found in the periphery adjacent to the area of CD138 expression. In vitro characterization of isolated CD138+ or SSEA‐1+ cell subpopulations revealed that CD138+ cells lose their retinal progenitor characteristics between E13 and E16, suggesting that they commit to becoming nonpigmented CE cells within this period. By in vivo mouse models, we found that stabilized β‐catenin expanded the area of CD138+ nonpigmented CE and that elimination of β‐catenin inhibited development of nonpigmented CE cells. These findings are the first to use cell‐surface markers to ascertain the spatial and temporal transitions that occur in developing CE.

In vitro cell subtype-specific transduction of adeno-associated virus in mouse and marmoset retinal explant culture.

Adeno-associated virus (AAV) is a non-pathogenic human parvovirus that can infect both non-proliferating and proliferating cells. Owing to its favorable safety profile, AAV is regarded as suitable for clinical purposes such as gene therapy. The target cell types of AAV depend largely on the serotype. In the retina, AAV has been used to introduce exogenous genes into photoreceptors, and photoreceptor-specific enhancers/promoters are used in most cases. Therefore, serotype specificity of AAV in retinal subtypes is unclear, particularly in vitro. We compared its infection profile in mouse and monkey retinas using EGFP under the control of the CAG promoter, which expressed the gene ubiquitously and strongly regardless of cell type. AAV1, 8, and 9 infected the horizontal cells when an embryonic day-17 retina was used as a host. Amacrine cell was also a major target of AAVs, and a small number of rod photoreceptors were infected. When adult retinas were used as a host, the main target of AAV was Müller glia. A small number of rod photoreceptors were also infected. In the adult common marmoset retina, rod and cone photoreceptors were efficiently infected by AAV1, 8, and 9. A portion of the Müller glia and amacrine cells were also infected. In summary, the infection specificity of different AAV serotypes did not differ, but was dependent on the stage of the host retina. In addition, infection specificities differed between mature marmoset retinas and mature mouse retinas.

β-Catenin signaling regulates the timing of cell differentiation in mouse retinal progenitor cells

Wnt signaling is important in development and carcinogenesis. We previously showed that active β-catenin or Lef-1 in the mammalian retinal culture prevents differentiation of retinal cells without modifying cellular proliferation. In this study, we investigated the in vivo role of β-catenin in mouse retinal differentiation in transgenic mice, in which retinal-specific activation or inactivation of β-catenin was achieved with Cre recombinase. The gain-of-function mice exhibited small eyes and large cell aggregates consisting of early progenitor cells labeled with SSEA-1 in the peripheral retina. In the loss-of-function mice, we observed a reduced number of SSEA-1-positive progenitor cells and the presence of differentiated cells in the β-catenin ablated retinal region. Interestingly, the number of proliferating cells in the β-catenin gain-of-function mice was highly downregulated, and the proliferation index detected by Ki67 expression was slightly lower than that of control mice in the β-catenin loss-of-function mice. The Gsk-3β inhibitor BIO induced expression of Id3, which was highly expressed in SSEA-1-positive cells, and transiently maintained SSEA-1-positive retinal progenitor cells (RPCs). Forced expression of Id3 in RPCs mimicked the effects of BIO. Taken together, β-catenin signaling regulates the timing of differentiation in RPCs by inhibiting premature differentiation of them partly through the regulation of Id3 expression.

DNA Methylation Is Involved in the Expression of miR-142-3p in Fibroblasts and Induced Pluripotent Stem Cells

MicroRNAs are differentially expressed in cells and regulate multiple biological processes. We have been analyzing comprehensive expression patterns of microRNA in human and mouse embryonic stem and induced pluripotent stem cells. We determined microRNAs specifically expressed in these pluripotent stem cells, and miR-142-3p is one of such microRNAs. miR-142-3p is expressed at higher levels in induced pluripotent stem cells relative to fibroblasts in mice. Level of expression of miR142-3p decreased during embryoid body formation from induced pluripotent stem cells. Loss-of-function analyses of miR-142-3p suggested that miR-142-3p plays roles in the proliferation and differentiation of induced pluripotent stem cells. CpG motifs were found in the 5′ genomic region of the miR-142-3p; they were highly methylated in fibroblasts, but not in undifferentiated induced pluripotent stem cells. Treating fibroblasts with 5-aza-2′-deoxycytidine increased the expression of miR-142-3p significantly and reduced methylation at the CpG sites, suggesting that the expression of miR-142-3p is suppressed by DNA methylation in fibroblasts. Luciferase analysis using various lengths of the 5′ genomic region of miR142-3p indicated that CpGs in the proximal enhancer region may play roles in suppressing the expression of miR-142-3p in fibroblasts.