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.

Light Stress-Induced Increase of Sphingosine 1-Phosphate in Photoreceptors and Its Relevance to Retinal Degeneration.

Sphingosine 1-phosphate (S1P) is a potent lipid mediator that modulates inflammation and angiogenesis. In this study, we investigated the possible involvement of S1P in the pathology of light-induced retinal degeneration in vivo and in vitro. The intracellular S1P and sphingosine kinase (SphK) activity in a photoreceptor cell line (661W cells) was significantly increased by exposure to light. The enhancement of SphK1 expression was dependent on illumination, and all-trans-retinal significantly promoted SphK1 expression. S1P treatment reduced protein kinase B (Akt) phosphorylation and increased the protein expression of cleaved caspase-3, and induced photoreceptor cell apoptosis. In vivo, light exposure enhanced the expression of SphK1 in the outer segments of photoreceptors. Intravitreal injection of a SphK inhibitor significantly suppressed the thinning of the outer nuclear layer and ameliorated the attenuation of the amplitudes of a-waves and b-waves of electroretinograms during light-induced retinal degeneration. These findings imply that light exposure induces the synthesis of S1P in photoreceptors by upregulating SphK1, which is facilitated by all-trans-retinal, causing retinal degeneration. Inhibition of this enhancement may be a therapeutic target of outer retinal degeneration, including age-related macular degeneration.

Foxr2 promotes formation of CNS-embryonal tumors in a Trp53-deficient background.

BACKGROUND: Embryonal tumors in the central nervous system (CNS) are primary, aggressive, and poorly differentiated pediatric brain tumors. We identified forkhead box R2 (Foxr2) as an oncogene for medulloblastoma through a transposon-based insertional mutagenesis screen. Foxr2 translocation has been identified in a subset of human embryonal tumors of the CNS, designated as CNS neuroblastoma with Foxr2 activation (CNS NB-Foxr2); however, the in vivo functions of Foxr2 remain elusive. METHODS: We analyzed the effect of Foxr2 overexpression in the mouse brain by generating a transgenic strain that expresses Foxr2 in the entire brain under a transformation related protein 53 (Trp53)-deficient background. We performed histological analysis of tumors and characterized tumor-derived sphere-forming cells. We investigated gene expression profiles of tumor-derived cells. RESULTS: Foxr2 and Trp53 loss promoted tumor formation in the olfactory bulb (OB) and brainstem (BS). The tumors showed the common morphological features of small round blue cell tumors, exhibiting divergent, mainly neuronal and glial, patterns of differentiation, which corresponds to the definition of CNS-embryonal tumors. Importantly, all mice developed CNS-embryonal tumors. In the OB, early proliferative lesions consisting of oligodendrocyte transcription factor 2 (Olig2+) cells were observed, indicating that Foxr2 expression expanded Olig2+ cells in the OB. Tumor-derived cells formed spheres in vitro and induced tumors that recapitulated the parental tumor upon transplantation, indicating the presence of tumor-initiating cells. Gene expression profiling revealed that OB and BS tumor cells were enriched for the expression of the genes specific to CNS NB-Foxr2. CONCLUSION: Our data demonstrate that Foxr2 plays a causative role in the formation of CNS-embryonal tumors.

Ras activation in retinal progenitor cells induces tumor formation in the eye.

The RAS gene family members, H-RAS, K-RAS, and N-RAS, are frequently mutated in human cancer. A subset of retinal tumors displays K-RAS mutations; however, the specific role of RAS activation on retinal tumor formation is unclear. To examine the role of RAS in retinal development, we overexpressed the mutant H-RAS gene (G12V) in retinal progenitor cells (RPCs), a multipotent progenitor cell population that gives rise to all six neuron types in the retina and to the Muller glia. The Msi1CreER mouse strain was used to induce mosaic activation of Ras (RasV12) in the RPCs of the postnatal retina. RAS-activated RPCs translocated to the basal part of the retina, differentiated into cells with glial characteristics, and underwent apoptosis. We next induced RAS activation in a large population of RPCs in the embryonic retina using the Pax6Cre mouse strain. In contrast to the phenotype observed in Msi1CreER;RasV12 mice, Ras-activated cells retained their apical attachment. Basal translocation was partially suppressed in the retina of Pax6Cre;RasV12 mice, indicating that basal translocation of Ras-activated cells was not cell autonomous. Notably, RAS-activated retinal cells were highly proliferative and promoted the formation of eye tumors in Pax6Cre;RasV12 mice. Together, our data indicate that the tumorigenicity of RAS activation in RPCs is context dependent, with tumor formation occurring when RAS activity is present in a large cluster of embryonic RPCs.

The role of Zhx2 transcription factor in bipolar cell differentiation during mouse retinal development.

We found that the Zhx2 gene (whose product is known to act as a tumor suppressor in hepatocellular carcinoma) is expressed in embryonic retinal progenitors and in developing cone bipolar cells in the postnatal retina, as well as in Müller glia in the mature retina. To examine the functions of Zhx2 protein during retinal development, we performed loss- and gain-of-function analyses using a retinal explant culture system. We introduced a plasmid encoding Zhx2 shRNA into isolated mouse retinas at E17.5, and the retinas were cultured as explants. After 3 days of culture, proliferation was slightly enhanced, leading to retinas thicker than in the control, but this phenomenon was observed only transiently. After 14 days of the culture, the thickness and gross morphology of retinas expressing sh-Zhx2 were indistinguishable from those of the control. The numbers of rod cells, amacrine cells, and Müller glia were the same in both groups. However, although the total number of bipolar cells was the same, the experimental group saw an increased population of ON bipolar cells, and decreased numbers of a subset of OFF bipolar cells. We also examined the effects of overexpression of Zhx2. Although Zhx2 acts as a tumor suppressor, its overexpression in developing retinas did not lead to any discernible difference in retinal thickness, suggesting that proliferation activity was not affected. After 14 days of explant culture, the total number of bipolar cells decreased, and subset composition was altered. Taken together, these results suggest that Zhx2 plays roles in the regulation of bipolar cell subset fate determination during retinal development.

Author Correction: Analysis of Müller glia specific genes and their histone modification using Hes1-promoter driven EGFP expressing mouse.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Sall1 Regulates Microglial Morphology Cell Autonomously in the Developing Retina.

Retinal degeneration often accompanies microglial activation and infiltration of monocyte-derived macrophages into the retina, resulting in the coexistence of microglia and monocyte-derived macrophages in the retina. We previously showed that the Sall1 zinc-finger transcriptional factor is expressed specifically in microglia within the retinal phagocyte pool, and analyses of Sall1 knockout mice revealed that microglial morphology changed from a ramified to a more amoeboid appearance in the developing retina. To investigate further whether Sall1 functions autonomously in microglia, we generated Sall1 conditional knockout mice, in which Sall1 was depleted specifically in the Cx3cr1+ microglial compartment of the developing retina. Sall1-deficient microglia exhibited morphological abnormalities on embryonic day 18 that strikingly resembled the phenotype observed in Sall1 knockout mice, demonstrating that Sall1 regulates microglial morphology cell autonomously. Analysis of the postnatal retina revealed that Sall1-deficient microglia extended their processes and their morphology became comparable to that of wild-type microglia on postnatal day 21, indicating that Sall1 is essential for microglial ramification in the developing retina, but not in the postnatal retina.

Spontaneous development of intratumoral heterogeneity in a transposon-induced mouse model of glioma.

Glioma is the most common form of malignant brain cancer in adults. The Sleeping Beauty (SB) transposon-based glioma mouse model allows for effective in vivo analysis of candidate genes. In the present study, we developed a transposon vector that encodes the triple combination of platelet-derived growth factor subunit A (PDGFA), and shRNAs against Nf1 and Trp53 (shNf1/shp53). Initiation and progression of glioma in the brain were monitored by expression of a fluorescent protein. Transduction of the vector into neural progenitor and stem cells (NPC) in the subventricular zone (SVZ) of the neonatal brain induced proliferation of oligodendrocyte precursor cells, and promoted formation of highly penetrant malignant gliomas within 2-4 months. Cells isolated from the tumors were capable of forming secondary tumors. Two transposon vectors, encoding either PDGFA or shNf1/shp53 were co-electroporated into NPC. Cells expressing PDGFA or shNf1/shp53 were labeled with unique fluorescent proteins allowing visualization of the spatial distribution of cells with different genetic alterations within the same tumor. Tumor cells located at the center of tumors expressed PDGFA at higher levels than those located at the periphery, indicating that intratumoral heterogeneity in PDGFA expression levels spontaneously developed within the same tumor. Tumor cells comprising the palisading necrosis strongly expressed PDGFA, suggesting that PDGFA signaling is involved in hypoxic responses in glioma. The transposon vectors developed are compatible with any genetically engineered mouse model, providing a useful tool for the functional analysis of candidate genes in glioma.

RNA helicase DHX15 acts as a tumour suppressor in glioma.

BACKGROUND: Glioblastoma is the most common form of malignant brain cancer and has a poor prognosis in adults. We identified Dhx15 as a candidate tumour suppressor gene in glioma by transposon-based mutagenesis. Dhx15 is an adenosine triphosphate (ATP)-dependent RNA helicase belonging to the DEAH-box (DHX) helicase family, but its role in cancer remains elusive. METHODS: DHX15 expression levels were examined in glioma cell lines. DHX15 functions were examined by gain- and loss-of-function analyses. Protein motifs required for the function of DHX15 were investigated by the analysis of mutant proteins. RESULTS: DHX15 expression was lower in human glioma cell lines than in normal neural stem cells. Dhx15 knockdown resulted in enhanced proliferation of primary immortalised mouse astrocytes, supporting the notion that DHX15 is a tumour suppressor. Retroviral-mediated transduction of DHX15 into glioma cell lines suppressed proliferation and foci formation in vitro. Moreover, DHX15 suppressed tumour formation in a xenograft mouse model. ATPase activity was not required for the growth-inhibitory function of DHX15; however, the Ia, Ib, IV, and V motifs, which act as RNA-binding domains in DHX15, were essential. qPCR analysis revealed that DHX15 suppressed expression of NF-κB downstream target genes as well as the genes involved in splicing. CONCLUSIONS: These findings provide evidence that DHX15 acts as a tumour suppressor gene in glioma.

Analysis of Müller glia specific genes and their histone modification using Hes1-promoter driven EGFP expressing mouse.

Retinal neurons and Müller glia are generated from a common population of multipotent retinal progenitor cells. We purposed to identify Müller glia-specific molecular signatures during retinal development. Using transgenic mice carrying the Hes1 promoter (pHes1) followed by EGFP, we purified EGFP-positive Müller glia and other EGFP-negative retinal cells from developing retinas and subjected them to RNA sequencing analysis. Gene expression pattern of EGFP-positive cell was similar to genes expressed in retinal progenitors, and they were downregulated in other cell lineages. Then, we examined the modification profiles of H3K27me3 and H3K4me3 by referring to chromatin immunoprecipitation-sequencing data of rods and other cells. Clustering of the H3K4me3 and H3K27me3 values followed by ontology analysis revealed a high incidence of transcription factors including Hes1 in clusters with high H3K27me3 levels. Hes1 expression level decreased dramatically, and the H3K27me3 level at the Hes1-locus was upregulated strongly during retinal development. Furthermore, the Hes1 expression level was upregulated in an Ezh2-knockout retina. These results suggest that downregulation of Müller glia-related genes in other lineage rather than upregulation of them in Müller glia contributed Müller-specific molecular features, and a role for modified H3K27me3 in suppressing Müller glia-related genes in other retinal cell lineages to avoid unfavorable expression.

Docosahexaenoic acid preserves visual function by maintaining correct disc morphology in retinal photoreceptor cells.

Docosahexaenoic acid (DHA) has essential roles in photoreceptor cells in the retina and is therefore crucial to healthy vision. Although the influence of dietary DHA on visual acuity is well known and the retina has an abundance of DHA-containing phospholipids (PL-DHA), the mechanisms associated with DHA's effects on visual function are unknown. We previously identified lysophosphatidic acid acyltransferase 3 (LPAAT3) as a PL-DHA biosynthetic enzyme. Here, using comprehensive phospholipid analyses and imaging mass spectroscopy, we found that LPAAT3 is expressed in the inner segment of photoreceptor cells and that PL-DHA disappears from the outer segment in the LPAAT3-knock-out mice. Dynamic light-scattering analysis of liposomes and molecular dynamics simulations revealed that the physical characteristics of DHA reduced membrane-bending rigidity. Following loss of PL-DHA, LPAAT3-knock-out mice exhibited abnormalities in the retinal layers, such as incomplete elongation of the outer segment and decreased thickness of the outer nuclear layers and impaired visual function, as well as disordered disc morphology in photoreceptor cells. Our results indicate that PL-DHA contributes to visual function by maintaining the disc shape in photoreceptor cells and that this is a function of DHA in the retina. This study thus provides the reason why DHA is required for visual acuity and may help inform approaches for overcoming retinal disorders associated with DHA deficiency or dysfunction.

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.

Role of transcription factor Tgif2 in photoreceptor differentiation in the mouse retina.

5'TG3'-interacting factors (TGIFs) function as transcriptional repressors. Defects in TGIFs cause severe abnormalities in the developing brain and face. We found that Tgif2 was highly expressed in the mouse retina at early stages of development and examined its role in retinal development. Knockdown of Tgif2 in retinal explants at E14 using shRNA (sh-Tgif2) resulted in a decreased number of rod photoreceptors, whereas the number of cone photoreceptors increased without perturbation of cell proliferation and apoptosis. Concomitantly, the expression levels of photoreceptor-related genes were decreased in sh-Tgif2-introduced retinal explants. To examine the in vivo effects of Tgif2 overexpression, we generated Tgif2 conditional knock-in mice (Tgif2cKI). Although retinal cell differentiation, based on the relative proportions of retinal subtypes in the mature retina, was not affected, we observed abnormal localization of cone photoreceptor cell nuclei in the outer nuclear layer of the Tgif2cKI retina. However, electrical retinography suggest that cones in Tgif2cKI were functionally equivalent to those of wild mice. Our study revealed that Tgif2 participates in photoreceptor cell differentiation in the early stages of retinal development and regulates proper subretinal localization of the cone photoreceptors.

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.

Identification of RNA-Binding Protein LARP4B as a Tumor Suppressor in Glioma.

Transposon-based insertional mutagenesis is a valuable method for conducting unbiased forward genetic screens to identify cancer genes in mice. We used this system to elucidate factors involved in the malignant transformation of neural stem cells into glioma-initiating cells. We identified an RNA-binding protein, La-related protein 4b (LARP4B), as a candidate tumor-suppressor gene in glioma. LARP4B expression was consistently decreased in human glioma stem cells and cell lines compared with normal neural stem cells. Moreover, heterozygous deletion of LARP4B was detected in nearly 80% of glioblastomas in The Cancer Genome Atlas database. LARP4B loss was also associated with low expression and poor patient survival. Overexpression of LARP4B in glioma cell lines strongly inhibited proliferation by inducing mitotic arrest and apoptosis in four of six lines as well as in two patient-derived glioma stem cell populations. The expression levels of CDKN1A and BAX were also upregulated upon LARP4B overexpression, and the growth-inhibitory effects were partially dependent on p53 (TP53) activity in cells expressing wild-type, but not mutant, p53. We further found that the La module, which is responsible for the RNA chaperone activity of LARP4B, was important for the growth-suppressive effect and was associated with BAX mRNA. Finally, LARP4B depletion in p53 and Nf1-deficient mouse primary astrocytes promoted cell proliferation and led to increased tumor size and invasiveness in xenograft and orthotopic models. These data provide strong evidence that LARP4B serves as a tumor-suppressor gene in glioma, encouraging further exploration of the RNA targets potentially involved in LARP4B-mediatd growth inhibition. Cancer Res; 76(8); 2254-64. ©2016 AACR.

Expression pattern of Ccr2 and Cx3cr1 in inherited retinal degeneration.

BACKGROUND: Though accumulating evidence suggests that microglia, resident macrophages in the retina, and bone marrow-derived macrophages can cause retinal inflammation which accelerates photoreceptor cell death, the details of how these cells are activated during retinal degeneration (RD) remain uncertain. Therefore, it is important to clarify which cells play a dominant role in fueling retinal inflammation. However, distinguishing between microglia and macrophages is difficult using conventional techniques such as cell markers (e.g., Iba-1). Recently, two mouse models for visualizing chemokine receptors were established, Cx3cr1 (GFP/GFP) and Ccr2 (RFP/RFP) mice. As Cx3cr1 is expressed in microglia and Ccr2 is reportedly expressed in activated macrophages, these mice have the potential to distinguish microglia and macrophages, yielding novel information about the activation of these inflammatory cells and their individual roles in retinal inflammation. METHODS: In this study, c-mer proto-oncogene tyrosine kinase (Mertk) (-/-) mice, which show photoreceptor cell death due to defective retinal pigment epithelium phagocytosis, were employed as an animal model of RD. Mertk (-/-) Cx3cr1 (GFP/+) Ccr2 (RFP/+) mice were established by breeding Mertk (-/-) , Cx3cr1 (GFP/GFP) , and Ccr2 (RFP/RFP) mice. The retinal morphology and pattern of inflammatory cell activation and invasion of Mertk (-/-) Cx3cr1 (GFP/+) Ccr2 (RFP/+) mice were evaluated using retina and retinal pigment epithelium (RPE) flat mounts, retinal sections, and flow cytometry. RESULTS: Four-week-old Mertk (-/-) Cx3cr1 (GFP/+) Ccr2 (RFP/+) mice showed Cx3cr1-GFP-positive microglia in the inner retina. Cx3cr1-GFP and Ccr2-RFP dual positive activated microglia were observed in the outer retina and subretinal space of 6- and 8-week-old animals. Ccr2-RFP single positive bone marrow-derived macrophages were observed to migrate into the retina of Mertk (-/-) Cx3cr1 (GFP/+) Ccr2 (RFP/+) mice. These invading cells were still observed in the subretinal space in 18-week-old animals. CONCLUSIONS: Cx3cr1-GFP-positive microglia and Ccr2-RFP-positive macrophages were distinguishable in the retinas of Mertk (-/-) Cx3cr1 (GFP/+) Ccr2 (RFP/+) mice. In addition, Ccr2 expression in Cx3cr1 positive microglia is a feature of microglial activation in RD. Mertk (-/-) Cx3cr1 (GFP/+) Ccr2 (RFP/+) mice enabled observation of microglial activation over time during RD and may be useful for developing inflammation-targeted treatment strategies for RD in the future.

Transposon mutagenesis identifies genes and evolutionary forces driving gastrointestinal tract tumor progression.

To provide a more comprehensive understanding of the genes and evolutionary forces driving colorectal cancer (CRC) progression, we performed Sleeping Beauty (SB) transposon mutagenesis screens in mice carrying sensitizing mutations in genes that act at different stages of tumor progression. This approach allowed us to identify a set of genes that appear to be highly relevant for CRC and to provide a better understanding of the evolutionary forces and systems properties of CRC. We also identified six genes driving malignant tumor progression and a new human CRC tumor-suppressor gene, ZNF292, that might also function in other types of cancer. Our comprehensive CRC data set provides a resource with which to develop new therapies for treating CRC.

Identification of FoxR2 as an oncogene in medulloblastoma.

Medulloblastoma is the most common pediatric brain tumor, and in ∼25% of cases, it is driven by aberrant activation of the Sonic Hedgehog (SHH) pathway in granule neuron precursor (GNP) cells. In this study, we identified novel medulloblastoma driver genes through a transposon mutagenesis screen in the developing brain of wild-type and Trp53 mutant mice. Twenty-six candidates were identified along with established driver genes such as Gli1 and Crebbp. The transcription factor FoxR2, the most frequent gene identified in the screen, is overexpressed in a small subset of human medulloblastoma of the SHH subtype. Tgif2 and Alx4, 2 new putative oncogenes identified in the screen, are strongly expressed in the SHH subtype of human medulloblastoma. Mutations in these two genes were mutually exclusive with mutations in Gli1 and tended to cooccur, consistent with involvement in the SHH pathway. Notably, Foxr2, Tgif2, and Alx4 activated Gli-binding sites in cooperation with Gli1, strengthening evidence that they function in SHH signaling. In support of an oncogenic function, Foxr2 overexpression transformed NIH3T3 cells and promoted proliferation of GNPs, the latter of which was also observed for Tgif2 and Alx4. These findings offer forward genetic and functional evidence associating Foxr2, Tgif2, and Alx4 with SHH subtype medulloblastoma.

Profiling of microRNA in human and mouse ES and iPS cells reveals overlapping but distinct microRNA expression patterns.

Using quantitative PCR-based miRNA arrays, we comprehensively analyzed the expression profiles of miRNAs in human and mouse embryonic stem (ES), induced pluripotent stem (iPS), and somatic cells. Immature pluripotent cells were purified using SSEA-1 or SSEA-4 and were used for miRNA profiling. Hierarchical clustering and consensus clustering by nonnegative matrix factorization showed two major clusters, human ES/iPS cells and other cell groups, as previously reported. Principal components analysis (PCA) to identify miRNAs that segregate in these two groups identified miR-187, 299-3p, 499-5p, 628-5p, and 888 as new miRNAs that specifically characterize human ES/iPS cells. Detailed direct comparisons of miRNA expression levels in human ES and iPS cells showed that several miRNAs included in the chromosome 19 miRNA cluster were more strongly expressed in iPS cells than in ES cells. Similar analysis was conducted with mouse ES/iPS cells and somatic cells, and several miRNAs that had not been reported to be expressed in mouse ES/iPS cells were suggested to be ES/iPS cell-specific miRNAs by PCA. Comparison of the average expression levels of miRNAs in ES/iPS cells in humans and mice showed quite similar expression patterns of human/mouse miRNAs. However, several mouse- or human-specific miRNAs are ranked as high expressers. Time course tracing of miRNA levels during embryoid body formation revealed drastic and different patterns of changes in their levels. In summary, our miRNA expression profiling encompassing human and mouse ES and iPS cells gave various perspectives in understanding the miRNA core regulatory networks regulating pluripotent cells characteristics.

Musashi1-CreER(T2) : a new cre line for conditional mutagenesis in neural stem cells.

The RNA-binding protein Musashi1 (Msi1) is one of two mammalian homologues of Drosophila Musashi, which is required for the asymmetric cell division of sensory organ precursor cells. In the mouse central nervous system (CNS), Msi1 is preferentially expressed in mitotically active progenitor cells in the ventricular zone (VZ) of the neural tube during embryonic development and in the subventricular zone (SVZ) of the postnatal brain. Previous studies showed that cells in the SVZ can contribute to long-term neurogenesis in the olfactory bulb (OB), but it remains unclear whether Msi1-expressing cells have self-renewing potential and can contribute to neurogenesis in the adult. Here, we describe the generation of Msi1-CreER(T2) knock-in mice and show by cell lineage tracing that Msi1-CreER(T2) -expressing cells mark neural stem cells (NSCs) in both the embryonic and adult brain. Msi1-CreER(T2) mice thus represent a new tool in our arsenal for genetically manipulating NSCs, which will be essential for understanding the molecular mechanisms underlying neural development.