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.

Sall1 plays pivotal roles for lens fiber cell differentiation in mouse.

Mammals possess four Sall transcription factors that play various roles in organogenesis. Previously, we found that Sall1 is expressed in microglia in the central nervous system, and it plays pivotal roles in microglia maturation. In the eye, Sall1 was also expressed in the developing lens, and we examined its role in lens development. A knock-in mouse harboring the EGFP gene in the Sall1 locus (Sall1-gfp) was used to analyze the Sall1 expression pattern. In Sall1-gfp/wild, EGFP was expressed throughout the presumptive lens at E11.5, and subsequently the expression in the lens epithelium became weaker. After birth, signals were observed in the equator region. The effects of Sall1 knockout on lens development were examined in Sall1-gfp/gfp. Lens sections revealed small vacuole-like holes and gaps in the center of the lens fibers at E14.5. Subsequently, the vacuoles appeared in most regions of the fiber cells. Electron microscopic analysis indicated that the vacuoles were between the fiber cells, leading to huge gaps. In addition, contact between the lens epithelium and apical end of the fiber cell was disrupted, and there were gaps between the adjoining lens epithelial cells. However, gap junction structure was observed by electron microscopic analysis, and immunostaining of Zo1 showed rather appropriate expression pattern. Immunohistochemistry indicated that the major lens transcription factors Prox1 and Pax6 were expressed in relatively normal patterns. However, although the expression of Prox1 and Pax6 decreased in nuclei in the control lens, it remained in Sall1-gfp/gfp. In addition, lower expression level of c-Maf protein was observed. Therefore, Sall1 is strongly expressed in the lens from the early developmental stage and plays an essential role in the maintenance of fiber cell and lens epithelium adhesion.

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.

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.

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.

MicroRNA-7a regulates Müller glia differentiation by attenuating Notch3 expression.

miRNA-7a plays critical roles in various biological aspects in health and disease. We aimed to reveal roles of miR-7a in mouse retinal development by loss- and gain-of-function analyses of miR-7a. Plasmids encoding miR-7a or miR-7a-decoy (anti-sense miR-7a) were introduced into mouse retina at P0, and the retina was cultured as explant. Then, proliferation of retinal progenitors and differentiation of retinal subtypes were examined by immunostaining. miR-7a had no apparent effect on the proliferation of retinal progenitor cells. However, the expression of Müller glia marker, cyclin D3, was reduced by miR-7a overexpression and up-regulated by miR-7a decoy, suggesting that miR-7a negatively regulates differentiation of Müller glia. Targets of miR-7a, which were predicted by using a public program miRNA.org, and Notch3 was suggested to be one of candidate genes of miR-7a target. Notch3 3' UTR appeared to contain complementary sequence to the seed sequence of miR-7a. A reporter assay in NIH3T3 cells using a plasmid containing multiple repeats of potential target sequence of 3' Notch UTR showed that miR-7a suppress expression of reporter EGFP through 3'UTR region. Expression of sh-Notch3 and over-expression of NICD3 in retina suggested that miR-7a regulates Müller glia differentiation through attenuation of Notch3 expression. Taken together, we revealed that the miR-7a regulates the differentiation of Müller glia through the suppression of Notch3 expression.

ß-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 ß-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 ß-catenin in mouse retinal differentiation in transgenic mice, in which retinal-specific activation or inactivation of ß-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 ß-catenin ablated retinal region. Interestingly, the number of proliferating cells in the ß-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 ß-catenin loss-of-function mice. The Gsk-3ß 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, ß-catenin signaling regulates the timing of differentiation in RPCs by inhibiting premature differentiation of them partly through the regulation of Id3 expression.

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.

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.

Apigenin Regulates Activation of Microglia and Counteracts Retinal Degeneration.

Purpose: Photoreceptor degeneration is a major cause of blindness. Microglia are known to play key roles in the pathogenesis and progression of neural degeneration. We examined the possible use of apigenin, which is a naturally occurring flavonoid, for the treatment of photoreceptor degeneration through regulation of microglial activities. Methods: As in vitro analyses, BV2 and MG5 mouse microglia cell lines were stimulated in the presence or absence of apigenin, and their activation profile was examined. In vivo study was done using rd1 photoreceptor degeneration model, and apigenin was administered by intravitreal injection, and pathological feature was examined. Results: Cell survival was not affected by apigenin in either BV2 and MG5. Apigenin suppressed lipopolysaccharide (LPS)-induced chemokine production in both BV2 and MG5 cells, but phagocytosis was suppressed in MG5 cells but not in BV2 cells. Apigenin inhibited LPS-induced M1 activation but could not drive microglia toward the M2 phenotype. Apigenin suppressed the expression of miR-155 in a dose-dependent manner. Furthermore, the Ets protein level was suppressed by treatment of BV2 cells with apigenin. When rd1 mice were treated with apigenin by intravitreal injection, the expression of inflammatory chemokines in the retina was reduced, and activation of microglia and Müller glia was suppressed. Furthermore, the thickness of the outer nuclear layer of the retina of rd1 mice was thicker in apigenin-treated retinas. Conclusions: Taken together, local administration of apigenin to the retina is a potential therapeutic treatment for photoreceptor degeneration, which involves downregulation of microglia in the retina when photoreceptors are damaged.

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 beta-catenin expanded the area of CD138+ nonpigmented CE and that elimination of beta-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.

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.

Forkhead Box Protein P1 Is Dispensable for Retina but Essential for Lens Development.

Purpose: Forkhead box protein P1 (Foxp1) is a transcriptional repressor expressed in many tissues. We identified Foxp1 as a highly expressed gene in retinal progenitor cells and investigated its roles during eye development. Methods: Mouse eyes with Foxp1 gain- or loss-of-function were established in vitro and in vivo. Results: Foxp1 overexpression in retinal progenitor cells resulted in reduced rod and increased cone photoreceptors. However, retina-specific knockout of Foxp1 was not associated with retinal differentiation abnormalities. Foxp1 was highly expressed in the lens during early development, and continued to be expressed in epithelial and cortical fiber cells until adulthood. At birth, analyses of Foxp1 lens-specific knockout (Foxp1-L-CKO) mice showed no gross morphologic changes in germinal or central epithelial cell compared to the controls. However, the numbers of proliferating and apoptotic cells were significantly increased in Foxp1-L-CKO mice. In addition, clear Y-structures were not observed in either the posterior or anterior sutures of the Foxp1-L-CKO lenses. Mature lenses of Foxp1-L-CKO mice were small and opaque. The fiber cell structure in the core and the cortical fiber cell columns were disturbed in Foxp1-L-CKO mice at postnatal day 14, potentially accounting for the opacity. In addition, epithelial cells were not aligned into columns along the transition zone in Foxp1-L-CKO mice. Taken together, these results suggest that Foxp1 has a role during lens growth in epithelial and differentiating fiber cells. Conclusions: Loss of Foxp1 results in loss of suture and fiber cell alignment, which eventually causes lens opacity, suggesting that Foxp1 has a key role in establishing cortical lens architecture.

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.

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.

BMP signaling participates in late phase differentiation of the retina, partly via upregulation of Hey2.

Bone morphogenetic protein (BMP) plays pivotal roles in early retinal development. However, its roles in the late phase of retinal development remain unclear. We found that BMP receptors and ligands were expressed in the postnatal mouse retina. Furthermore, immunostaining revealed that phosphorylated Smads were enriched in various cells types in the inner nuclear layer postnatally. However, phosphorylated Smads were not detected in photoreceptors, suggesting that BMP may play roles in retinal cells in the inner nuclear layer. Forced expression of constitutively active BMP receptors during retinal development resulted in an increased number of bipolar cells and Müller glia and a decreased number of rod photoreceptors; however, proliferation was not perturbed. The expression of dominant negative BMP receptors resulted in a decreased number of Müller glia and bipolar cells. In addition, inhibiting BMP signaling in retinal monolayer cultures abrogated Müller glial process extension, suggesting that BMP signaling also plays a role in the maturation of Müller glia. The expression of the basic helix-loop-helix transcription factor Hey2 was induced by BMP signaling in retinas. The coexpression of sh-Hey2 with constitutively active BMP receptors suggested that the effects of BMP signaling on retinal differentiation could be attributed partly to the induction of Hey2 by BMP. We propose that BMP signaling plays pivotal roles in the differentiation of retinal progenitor cells into late differentiating retinal cell types and in the maturation of Müller glia; these effects were mediated, at least in part, by Hey2.

Critical roles of DNase1l3l in lens nuclear degeneration in zebrafish.

The vertebrate lens undergoes organelle and nuclear degradation during lens development, allowing the lens to become transparent. DNase2b is an enzyme responsible for nuclear degradation in the mouse lens; however, dnase2b expression in zebrafish showed a distribution pattern that differed from that in mice. No zebrafish dnase2b was detected by reverse-transcription polymerase chain reaction until around 120 h postfertilization (hpf), suggesting that dnase2b is not expressed in the critical period for lens nuclear degradation, which corresponds to 56-74 hpf. However, public database searches have indicated that dnase1l3l is strongly and specifically expressed in embryonic zebrafish lens. Whole mount in situ hybridization showed that dnase1l3l expression began around 36 hpf and was found exclusively in the lens until the adult stage. Morpholino (MO)-dependent downregulation of dnase1l3l expression during early development in zebrafish led to the failure of nuclear degradation in the lens. Immunostaining of lens sections showed that expression of Pax6, Prox1 and ß-catenin was comparable to the control in the early stage of development in dnase1l3l-MO injected embryos. However, downregulation of expression of these genes in lens was not observed in dnase1l3l-MO-treated zebrafish at 72 hpf, suggesting that the lens development was halted. Taken together, we showed that dnase1l3l plays major roles in nuclear degradation in zebrafish lens development. No homologous gene was found in other species in public databases, suggesting that dnase1l3l developed and acquired its function specifically in zebrafish.

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.

Robust Long-term Transduction of Common Marmoset Neuromuscular Tissue With rAAV1 and rAAV9.

Profiles of recombinant adeno-associated virus (rAAV)-mediated transduction show interspecies differences for each AAV serotype. Robust long-term transgene expression is generally observed in rodents, whereas insufficient transduction is seen in animals with more advanced immune systems. Non-human primates, including the common marmoset, could provide appropriate models for neuromuscular diseases because of their higher brain functions and physiological resemblance to humans. Strategies to induce pathologies in the neuromuscular tissues of non-human primates by rAAV-mediated transduction are promising; however, transgene expression patterns with rAAV transduction have not been elucidated in marmosets. In this study, transduction of adult marmoset skeletal muscle with rAAV9 led to robust and persistent enhanced green fluorescent protein (EGFP) expression that was independent of the muscle fiber type, although lymphocyte infiltration was recognized. Systemic rAAV injection into pregnant marmosets led to transplacental fetal transduction. Surprisingly, the intraperitoneal injection of rAAV1 and rAAV9 into the neonatal marmoset resulted in systemic transduction and persistent transgene expression without lymphocyte infiltration. Skeletal and cardiac muscle were effectively transduced with rAAV1 and rAAV9, respectively. Interestingly, rAAV9 transduction led to intense EGFP signaling in the axons of the corpus callosum. These transduction protocols with rAAV will be useful for investigating gene functions in the neuromuscular tissues and developing gene therapy strategies.Molecular Therapy-Nucleic Acids (2013) 2, e95; doi:10.1038/mtna.2013.21; published online 28 May 2013.

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.