Yasuo Ouchi

Specification of the Retinal Fate of Mouse Embryonic Stem Cells by Ectopic Expression of Rx/rax, a Homeobox Gene

ABSTRACT With the goal of generating retinal cells from mouse embryonic stem (ES) cells by exogenous gene transfer, we introduced the Rx/rax transcription factor, which is expressed in immature retinal cells, into feeder-free mouse ES cells (CCE). CCE cells expressing Rx/rax as well as enhanced green fluorescent protein (CCE-RX/E cells) proliferated and remained in the undifferentiated state in the presence of leukemia inhibitory factor, as did parental ES cells. We made use of mouse embryo retinal explant cultures to address the differentiation ability of grafted ES cells. Dissociated embryoid bodies were treated with retinoic acid for use as donor cells and cocultured with retina explants for 2 weeks. In contrast to the parental CCE cells, which could not migrate into host retinal cultures, CCE-RX/E cells migrated into the host retina and extended their process-like structures between the host retinal cells. Most of the grafted CCE-RX/E cells became located in the ganglion cell and inner plexiform layers and expressed ganglion and horizontal cell markers. Furthermore, these grafted cells had the electrophysiological properties expected of ganglion cells. Our data thus suggest that subpopulations of retinal neurons can be generated in retinal explant cultures from grafted mouse ES cells ectopically expressing the transcription factor Rx/rax.

Reduced Adult Hippocampal Neurogenesis and Working Memory Deficits in the Dgcr8-Deficient Mouse Model of 22q11.2 Deletion-Associated Schizophrenia Can Be Rescued by IGF2

DiGeorge syndrome chromosomal region 8 (Dgcr8), a candidate gene for 22q11.2 deletion-associated schizophrenia, encodes an essential component for microRNA (miRNA) biosynthesis that plays a pivotal role in hippocampal learning and memory. Adult neurogenesis is known to be important in hippocampus-dependent memory, but the role and molecular mechanisms of adult neurogenesis in schizophrenia remain unclear. Here, we show that Dgcr8 heterozygosity in mice leads to reduced cell proliferation and neurogenesis in adult hippocampus, as well as impaired hippocampus-dependent learning. Several schizophrenia-associated genes were downregulated in the hippocampus of Dgcr8+/− mice, and one of them, insulin-like growth factor 2 (Igf2), rescued the proliferation of adult neural stem cells both in vitro and in vivo. Furthermore, IGF2 improved the spatial working memory deficits in Dgcr8+/− mice. These data suggest that defective adult neurogenesis contributes to the cognitive impairment observed in 22q11.2 deletion-associated schizophrenia and could be rectified by IGF2.

Sox2 plays a role in the induction of amacrine and Müller glial cells in mouse retinal progenitor cells.

PURPOSE The transcription factor Sox2 plays important roles in both human and mouse retinal development. Although loss-of-function mutations in Sox2 have been studied in mice, gain-of-function experiments in the neural retina have been lacking. METHODS The detailed expression pattern of Sox2 in the developing mouse retina was examined by immunohistochemistry. Then, Sox2 was expressed in a retinal explant culture prepared from E17 mouse embryos by retrovirus-mediated gene transfer to examine its role in retinal development. In addition, shRNA was used to suppress Sox2 in a retinal explant using a retrovirus-mediated system. RESULTS Sox2 was expressed throughout the neuroblastic layer in the embryonic retina, but only in the inner nuclear layer in the mature retina. Double immunostaining revealed that Sox2 was expressed in Müller glial cells and in a subset of amacrine cells. Forced expression of Sox2 in a mouse retinal explant culture resulted in the dramatic accumulation of amacrine cells in the inner nuclear layer; in addition, cells expressing amacrine cell markers were also found on the innermost side of the outer nuclear layer. The expression of Pax6, which plays an important role in amacrine cell differentiation, was observed in the Sox2-expressing cells, and Sox2 activated the Pax6 promoter to drive luciferase expression in Y79 cells. A decrease in retinal progenitor cell proliferation accompanied these effects. The suppression of Sox2 expression by shRNA resulted in a decreased number of cells in the inner nuclear layer. CONCLUSIONS Therefore, ectopic Sox2 expression can induce amacrine cells in the mouse retina from stage E17 onward, possibly by facilitating cell cycle exit.

Negative regulation of retinal-neurite extension by beta-catenin signaling pathway.

Although there have been many studies on the regulation of neurite extension in mouse brain, such a mechanism in neural retina has remained to be clarified. To delineate the role of Wnt signaling in retinal development, we used a retrovirus-vector-mediated expression system to express various mutants forms of Wnt signaling members in E17.5 mouse retinal explant cultures, which are an excellent system to examine retinal development in vitro. Expression of constitutively active β-catenin or Lef-1 in the retinal cells resulted in failure of neurite extension, suggesting that β-catenin negatively regulates neurite extension in the retina through Lef-1 transcriptional activity. However, proliferation and differentiation of retinal cells into mature retinal cells such as rod-photoreceptor cells and Muller glia cells were not affected by perturbation of the Wnt-Lef-1 pathway. As in retinal cells, activation of β-catenin-Lef-1 signaling inhibited NGF-induced neurite extension in PC12 cells without affecting their proliferation. Interestingly, the Wnt-Lef-1 signaling pathway suppressed neurite extension without affecting Mek-1 signal activity, which is known to promote neurite extension. We found that MAPK was activated in retinal explant cultures, but that perturbation of MAPK signals did not affect neurite extension. Taken together, our data suggest that the Wnt pathway functions in proper neurite extension by opposing positive signals for promotion of neurite extension that are distinct from those of the MAPK pathway.

Forced Expression of miR-143 Represses ERK5/c-Myc and p68/p72 Signaling in Concert with miR-145 in Gut Tumors of ApcMin Mice

Recently, miR-143 and miR-145 have been shown to belong to a subset of microRNAs whose expression is controlled by a complex of a tumor suppressor p53 and DEAD-box RNA helicase subunits p68/p72. While accumulating studies have acknowledged that both miRNAs function as tumor suppressors and are similarly regulated, evidence of their coordinated action against tumorigenesis has been poorly presented. Herein, we establish transgenic mice that express miR-143 under the control of the CAG regulatory unit. When crossbred with ApcMin/+ mice, the development of tumors in the small intestines is significantly attenuated. In the transgenic small intestine tumors, the endogenous miR-145 is also enhanced and the expression of c-Myc and p68/p72, both of which have been reported to be pivotal for gut tumor development, is suppressed, corresponding to the downregulation of ERK5. We demonstrate that the combination of miR-143 and miR-145 inhibits the expression of c-Myc in human colon cancer cells, whereas miR-145 retards that of p72. Moreover, we show the possibilities that miR-145 modulates p72 expression through its 3′ untranslated region and that c-Myc downregulation is involved in both p68 suppression and miR-145 induction. These findings suggest that forced expression of miR-143, probably interacting with endogenous miR-145, inhibits ERK5/c-Myc and p68/p72/β-catenin signaling and hampers small intestine tumor development in ApcMin/+ mice. This unique cascade, in turn, may prevent overproduction of a subset of tumor suppressive miRNAs by repressing their own modulators, p68/p72.

The Heterochronic Genes lin-28a and lin-28b Play an Essential and Evolutionarily Conserved Role in Early Zebrafish Development

The Caenorhabditis elegans heterochronic gene pathway, which consists of a set of regulatory genes, plays an important regulatory role in the timing of stage-specific cell lineage development in nematodes. Research into the heterochronic gene pathway gave rise to landmark microRNA (miRNA) studies and showed that these genes are important in stem cell and cancer biology; however, their functions in vertebrate development are largely unknown. To elucidate the function of the heterochronic gene pathway during vertebrate development, we cloned the zebrafish homologs of the C. elegans let-7 miRNA-binding protein, Lin-28, and analyzed their function in zebrafish development. The zebrafish genome contains two Lin28-related genes, lin-28a and lin-28b. Similar to mammalian Lin28 proteins, both zebrafish Lin-28a and Lin-28b have a conserved cold-shock domain and a pair of CCHC zinc finger domains, and are ubiquitously expressed during early embryonic development. In a reciprocal fashion, the expression of downstream heterochronic genes, let-7 and lin-4/miR-125 miRNA, occurred subsequent to lin-28 expression. The knockdown of Lin-28a or Lin-28b function by morpholino microinjection into embryos resulted in severe cell proliferation defects during early morphogenesis. We found that the expression of let-7 miRNA was upregulated and its downstream target gene, lin-41, was downregulated in these embryos. Interestingly, the expression of miR-430, a key regulator of maternal mRNA decay, was downregulated in lin-28a and lin-28b morphant embryos, suggesting a role for Lin-28 in the maternal-to-zygotic transition in zebrafish. Taken together, our results suggest an evolutionarily conserved and pivotal role of the heterochronic gene pathway in early vertebrate embryogenesis.

Emerging evidence of insulin-like growth factor 2 as a memory enhancer: a unique animal model of cognitive dysfunction with impaired adult neurogenesis

Abstract In the current aging society, cognitive dysfunction is one of the most serious issues that should be urgently resolved. It also affects a wide range of age groups harboring neurological and psychiatric disorders, such as Alzheimer’s disease and schizophrenia. Although the molecular mechanism of memory impairment still remains to be determined, neuronal loss and dysfunction has been revealed to mainly attribute to its pathology. The discovery of neural stem cells in the adult brain that are proliferating and able to generate functional neurons has given rise to the idea that neuronal loss could be rescued by manipulating endogenous neural progenitor and stem cells. To this end, we must characterize them in detail and their developmental programming must be better understood. A growing body of evidence has indicated that insulin-like peptides are involved in learning and memory and maintenance of neural progenitor and stem cells, and clinical trials of insulin as a memory enhancer have begun. In contrast to the expectation of insulin and IGF1, the roles of IGF2 in cognitive ability have been poorly understood. However, recent evidence demonstrated in rodents suggests that IGF2 may play a pivotal role in adult neurogenesis and cognitive function. Here, we would like to review the rapidly growing world of IGF2 in cognitive neuroscience and introduce the evidence that its deficit is indeed involved in the impairment of the hippocampal neurogenesis and cognitive dysfunction in the model mouse of 22q11.2 deletion syndrome, which deletes Dgcr8, a critical gene for microRNA processing.

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.

Upregulation of Mir-21 Levels in the Vitreous Humor Is Associated with Development of Proliferative Vitreoretinal Disease

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by post-transcriptional inhibition of mRNA translation. Dysregulation of miRNAs, including circulating miRNAs, has been reported to play an important role in the development of various diseases, including fibrotic diseases. Aberrant expression of miRNAs in the vitreous humor of vitreoretinal diseased eyes has been reported. However, the expression pattern of miRNAs present in the vitreous humor of proliferative vitreoretinal disease (PVD) patients, including proliferative diabetic retinopathy (PDR), and proliferative vitreoretinopathy (PVR), remains unknown. To investigate the factors important for the development of PVD, we characterized the miRNAs present in the vitreous humor of PVD patients and analyzed the expression profiles of 377 miRNAs using quantitative polymerase chain reaction-based miRNA arrays. The expression of a specific subset of miRNAs, previously reported to be associated with the development of angiogenesis and fibrosis, was significantly altered in the vitreous of PVD patients. Among these miRNAs, we identified miR-21 as a candidate fibrotic miRNA with an important role in the pathogenesis of PVD. Increased miR-21 levels in the vitreous were associated with retinal fibrosis, including PVR and PDR. Because epithelial-mesenchymal transition (EMT) of retinal pigment epithelial cells (RPECs) plays a critical role in retinal fibrosis, the expression of miR-21 in human RPECs was determined. Its expression in RPECs was induced by transforming growth factor-β, a key growth factor involved in fibrogenesis, and was enhanced by high glucose culture conditions, suggesting that miR-21 expression positively correlates with disease progression. Gain- and loss-of-function studies revealed that miR-21 promoted cell proliferation and migration of ARPE-19 cells without affecting EMT-related gene expression. Together, our studies have identified miR-21 as a potential disease-modifying miRNA in the vitreous humor that is involved in the development of retinal fibrosis and may be a novel marker of PVD.