Musashi1 (Msi1) is an RNA-binding protein that is highly expressed in neural progenitor cells, including neural stem cells. In this study, the RNA-binding sequences for Msi1 were determined by in vitro selection using a pool of degenerate 50-mer sequences. All of the selected RNA species contained repeats of (G/A)U n AGU (n ‫؍‬ 1 to 3) sequences which were essential for Msi1 binding. These consensus elements were identified in some neural mRNAs. One of these, mammalian numb (m-numb), which encodes a membrane-associated antagonist of Notch signaling, is a likely target of Msi1. Msi1 protein binds in vitro-transcribed m-numb RNA in its 3-untranslated region (UTR) and binds endogenous m-numb mRNA in vivo, as shown by affinity precipitation followed by reverse transcription-PCR. Furthermore, adenovirus-induced Msi1 expression resulted in the down-regulation of endogenous m-Numb protein expression. Reporter assays using a chimeric mRNA that combined luciferase and the 3-UTR of m-numb demonstrated that Msi1 decreased the reporter activity without altering the reporter mRNA level. Thus, our results suggested that Msi1 could regulate the expression of its target gene at the translational level. Furthermore, we found that Notch signaling activity was increased by Msi1 expression in connection with the posttranscriptional down-regulation of the m-numb gene.

Homologues of the Musashi family of RNA-binding proteins are evolutionarily conserved across species. In mammals, two members of this family, Musashi1 (Msi1) and Musashi2 (Msi2), are strongly coexpressed in neural precursor cells, including CNS stem cells. To address the in vivo roles of msi in neural development, we generated mice with a targeted disruption of the gene encoding Msi1. Homozygous newborn mice frequently developed obstructive hydrocephalus with aberrant proliferation of ependymal cells in a restricted area surrounding the Sylvius aqueduct. These observations indicate a vital role for msi1 in the normal development of this subpopulation of ependymal cells, which has been speculated to be a source of postnatal CNS stem cells. On the other hand, histological examination and an in vitro neurosphere assay showed that neither the embryonic CNS development nor the self-renewal activity of CNS stem cells in embryonic forebrains appeared to be affected by the disruption of msi1, but the diversity of the cell types produced by the stem cells was moderately reduced by the msi1 deficiency. Therefore, we performed antisense ablation experiments to target both msi1 and msi2 in embryonic neural precursor cells. Administration of the antisense peptidenucleotides, which were designed to specifically down-regulate msi2 expression, to msi1 ؊/؊ CNS stem cell cultures drastically suppressed the formation of neurospheres in a dose-dependent manner. Antisense-treated msi1 ؊/؊ CNS stem cells showed a reduced proliferative activity. These data suggest that msi1 and msi2 are cooperatively involved in the proliferation and maintenance of CNS stem cell populations.

ABSTRACT

SummaryThe entire nucleotide sequence of the transfer region of IncI1 plasmid R64 was determined together with previously reported sequences. Twenty-two transfer genes, traE±Y and nuc, were newly identified in the present study. The protein products of 17 genes were detected by maxicell experiments or by the T7 RNA polymerase expression system. Mutagenesis experiments indicated that 16 genes were indispensable for R64 transfer both in liquid and on surfaces. In summary, the R64 transfer region located within an < 54 kb DNA segment was shown to encode the most complex transfer system so far studied. It contains at least 49 genes and may produce 58 different proteins as a result of shufflon DNA rearrangement and overlapping genes. Among the 49 genes, 23 tra, trb and nik genes have been shown to be indispensable for R64 conjugal transfer in liquid and on surfaces. Twelve additional pil genes are required only for liquid matings. The amino acid sequences of 10 R64 tra/trb products share similarity with those of the icm/dot products of Legionella pneumophila that are responsible for its virulence, suggesting that the R64 transfer and L. pneumophila icm/dot systems have evolved from a common ancestral genetic system.

FGFRs (®broblast growth factor receptors) are encoded by four genes (FGFR1 ± 4). Alternative splicing results in various receptor isoforms. The FGFR2-IIIb variant is present in a wide variety of epithelia, including the bladder epithelium. Recently, we have shown that FGFR2-IIIb is downregulated in a subset of transitional cell carcinomas of the bladder, and that this downregulation is associated with a poor prognosis. We investigated possible tumour suppressive properties of FGFR2-IIIb by transfecting two human bladder tumour cell lines, J82 and T24, which have no endogenous FGFR2-IIIb expression, with FGFR2-IIIb cDNA. No stable clones expressing FGFR2-IIIb were isolated with the J82 cell line. For the T24 cell line, stable transfectants expressing FGFR2-IIIb had reduced growth in vitro and formed fewer tumours in nude mice which, in addition, grew more slowly. The potential mechanisms leading to decreased FGFR2-IIIb mRNA levels were also investigated. The 5' region of the human FGFR2 gene was isolated and found to contain a CpG island which was partially methylated in more than half the cell lines and tumours which do not express FGFR2-IIIb. No homozygous deletion was identi®ed in any of the tumours or cell lines with reduced levels of FGFR2-IIIb. Mutational analysis of the entire coding region of FGFR2-IIIb at the transcript level was performed in 33 bladder tumours. In addition to normal FGFR2-IIIb mRNA, abnormal transcripts were detected in two tumour samples. These abnormal mRNAs resulted from exon skipping which aected the region encoding the kinase domain. Altogether, these results show that FGFR2-IIIb has tumour growth suppressive properties in bladder carcinomas and suggest possible mechanisms of FGFR2 gene inactivation.

Notch1 plays various important roles including the maintenance of the stem cell state as well as the promotion of glial fates in mammalian CNS development. However, because of the very low amount of the activated form of Notch1 present in vivo, its precise activation pattern has remained unknown. In this study, we mapped the active state of this signaling pathway in situ in the developing mouse brain using a specific antibody that recognizes the processed form of the intracellular domain of Notch1 cleaved by presenilin/c-secretase activity. By using this antibody, active state of Notch1 came to be detectable with a higher sensitivity than using conventional antibody against Notch1. We found that activated Notch1 was mainly detected in the nuclei of a subpopulation of radial glial cells, the majority of proliferating precursor cells in the ventricular zone (VZ). However, Notch1 activation was not detected in neuronal precursor cells positive for neuronal basic helix-loop-helix proteins or in differentiating neurons in the embryonic forebrain. Interestingly, we found that Notch1 was transiently activated in the astrocytic lineage during perinatal CNS development. Taken together, the present method has enabled us to determine the timing, gradients, and boundaries of the activation of Notch signaling.

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BackgroundRetinitis pigmentosa (RP) is an inherited human retinal disorder that causes progressive photoreceptor cell loss, leading to severe vision impairment or blindness. However, no effective therapy has been established to date. Although genetic mutations have been identified, the available clinical data are not always sufficient to elucidate the roles of these mutations in disease pathogenesis, a situation that is partially due to differences in genetic backgrounds.ResultsWe generated induced pluripotent stem cells (iPSCs) from an RP patient carrying a rhodopsin mutation (E181K). Using helper-dependent adenoviral vector (HDAdV) gene transfer, the mutation was corrected in the patient’s iPSCs and also introduced into control iPSCs. The cells were then subjected to retinal differentiation; the resulting rod photoreceptor cells were labeled with an Nrl promoter-driven enhanced green fluorescent protein (EGFP)-carrying adenovirus and purified using flow cytometry after 5 weeks of culture. Using this approach, we found a reduced survival rate in the photoreceptor cells with the E181K mutation, which was correlated with the increased expression of endoplasmic reticulum (ER) stress and apoptotic markers. The screening of therapeutic reagents showed that rapamycin, PP242, AICAR, NQDI-1, and salubrinal promoted the survival of the patient’s iPSC-derived photoreceptor cells, with a concomitant reduction in markers of ER stress and apoptosis. Additionally, autophagy markers were found to be correlated with ER stress, suggesting that autophagy was reduced by suppressing ER stress-induced apoptotic changes.ConclusionThe use of RP patient-derived iPSCs combined with genome editing provided a versatile cellular system with which to define the roles of genetic mutations in isogenic iPSCs with or without mutation and also provided a system that can be used to explore candidate therapeutic approaches.