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.

Candidate mammalian odorant receptors were first cloned some 6 years ago. The physiological function of these receptors in initiating transduction in olfactory receptor neurons remains to be established. Here, a recombinant adenovirus was used to drive expression of a particular receptor gene in an increased number of sensory neurons in the rat olfactory epithelium. Electrophysiological recording showed that increased expression of a single gene led to greater sensitivity to a small subset of odorants.

We report a new set of ultra-sensitive Ca 2+ indicators, yellow cameleon-Nano (YC-Nano), developed by engineering the Ca 2+ -sensing domain of a genetically encoded Ca 2+ indicator, YC2.60 or YC3.60. Their high Ca 2+ affinities (K d = 15-140 nM) and large signal change (1,450%) enabled detection of subtle Ca 2+ transients associated with intercellular signaling dynamics and neuronal activity, even in 100,000-cell networks. These indicators will be useful for studying information processing in living multi-cellular networks.3To decipher the principles of information processing in multi-cellular networks, such as a brain or developing embryo, it is essential to record cellular activity with fine We therefore generated YCs with a longer linker, containing 3 to 8 amino acids (designated 3 to 8) (Fig. 1a). The Ca 2+ affinity gradually increased as the linker was elongated and we named the resulting series of sensors, high-affinity yellow cameleons or "YC-Nano". For the 3 (Gly-Gly-Ser) linker the K d was 50 nM (YC-Nano50) and for the 4 (Gly-Gly-Gly-Ser) linker the K d was 30 nM (YC-Nano30). YCs with a 5 to 8 5 linker also had a higher affinity than YC2.60. The lowest K d (= 15 nM) was achieved with a 5 linker (Gly-Gly-Gly-Gly-Ser) (YC-Nano15); this indicator had the highest affinity of any GECI reported so far 11 (Fig. 1b, Supplementary Table 1 and Supplementary Fig. 1). Linker elongation was also effective for YC3.60, yielding YC-Nano140 (4; Gly-Gly-Gly-Ser, K d = 140 nM) and YC-Nano65 (5;Gly-Gly-Gly-Gly-Ser, K d = 65 nM) (Fig. 1b, Supplementary Fig. 1 and Supplementary Table 1).Kinetic measurement by stopped-flow fluorometry of YC-Nano140 and YC3.60 revealed that only the rate constant for the on reaction was increased, while that of the off reaction remained unchanged (Supplementary Fig. 2 Fig. 3). Supplementary Fig. 4). To verify the advantages of YC-Stimulation with 10 M cAMP yielded large FRET signal changes, assessed by ratiometric wide-field imaging of aggregation-competent cells (Fig. 1c). The YFP/CFP for the YC-Nano15-and YC2.60-expressing cells changed from 5.0 to 9.5 (R = 4.5) and 2.0 to 6.2 (R = 4.2), respectively. We next determined the amplitude of the Ca The increased signal strength achieved by optimizing the K d also allowed us to perform Ca 2+ imaging on a large spatial scale. The field of view for imaging could be expanded to a millimeter-sized network that included 100,000-Dictyostelium cells, in which the aggregation wave was clearly visible as a rotating spiral (Supplementary Fig. 5, Supplementary Video 2, R Nano15 = 0.6), indicating that YC-Nano15, unlike YC2.60, was useful for detecting multi-cellular network activity in self-organized signaling dynamics (R YC2.60 = 0.15, Supplementary Fig. 6, Supplementary Video 3).We next tested the performance of YC-Nano in a neuronal system (Supplementary Note 3 and Supplementary Figure 7). For this, we examined YC-Nano's sensitivity for the subtle Ca 2+ transient triggered by a single action potential (AP). YC-Nano15 and 7 YC3...

The adult cerebellum is functionally compartmentalized into clusters along the mediolateral axis (M-L clusters), and a variety of molecular makers are expressed in specific subsets of M-L clusters. These M-L clusters appear to be the basic structure in which cerebellar functions are performed, but the mechanisms by which cerebellar mediolateral compartmentalization is established are still unclear. To address these questions, we examined the development of M-L clusters using replication-defective adenoviral vectors. The adenoviral vectors effectively introduced foreign genes into the neuronal progenitor cells of the cerebellum in a birth date-specific manner, allowing us to observe the native behavior of each cohort of birth date-related progenitor cells. When the adenoviral vectors were injected into the midbrain ventricle of mouse embryos on embryonic days 10.5 (E10.5), E11.5, and E12.5, the virally infected cerebellar progenitor cells developed into Purkinje cells. Notably, the Purkinje cells that shared the same birth date formed specific subsets of M-L clusters in the cerebellum. Each subset of M-L clusters displayed nested and, in part, mutually complementary patterns, and these patterns were unchanged from the late embryonic stage to adulthood, suggesting that Purkinje cell progenitors are fated to form specific subsets of M-L clusters after their birth between E10.5 and E12.5. This study represents the first such direct observation of Purkinje cell development. Moreover, we also show that there is a correlation between the M-L clusters established by the birth date-related Purkinje cells and the domains of engrailed-2, Wnt-7B, L7/pcp2, and EphA4 receptor tyrosine kinase expression.

3.

Neural stem and precursor cells reside in the ventricular lining of the fetal forebrain, and may provide a cellular substrate for brain repair. To selectively identify and extract these cells, we infected dissociated fetal human brain cells with adenoviruses bearing the gene for green fluorescence protein (GFP), placed under the control of enhancer/promoters for two genes (nestin and musashi1) that are expressed in uncommitted neuroepithelial cells. The cells were then sorted by fluorescence-activated cell sorting (FACS) on the basis of E/nestin- or P/musashi1-driven GFP expression. Both P/musashi1:hGFP- and E/nestin:EGFP-sorted cells were multipotent: limiting dilution with clonal expansion as neurospheres, in tandem with retroviral lineage analysis and xenograft to E17 and P0-2 rat forebrain, revealed that each phenotype was able to both self-renew and co-generate neurons and glia. Thus, fluorescent genes placed under the control of early neural promoters allow neural stem cells to be specifically targeted, isolated, and substantially enriched from the fetal human brain.

These results demonstrated that the surgical outcome of ASF was superior to the surgical outcome of laminoplasty. Elderly patients treated with laminoplasty showed an especially poor surgical outcome. We suggest that hypermobility of vertebrae at the cord compression level is a risk factor for poor surgical outcome after laminoplasty. Based on these results, we recommend that ASF should be the first choice of treatment for patients with significant ossification of the posterior longitudinal ligament and a hypermobile cervical spine. When laminoplasty is used for such cases, the addition of posterior instrumented fusion would be desirable for stabilizing the spine and decreasing damage to the spinal cord.

To clarify the mechanism of cerebellar development, we have cloned a gene, named zic, encoding a zinc finger protein that is expressed abundantly in granule cells throughout development of the cerebellum. zic has a significant homology to the zinc finger domain of the Caenorhabditis elegans tra1 gene, the Drosophila cubitus interruptus Dominant gene, and the human GLI oncogene. An in situ hybridization study revealed that zic showed a restricted expression pattern in the granule cells and their putative precursor cells. It is also expressed at an early embryonic stage in the dorsal half of the neural tube. The expression pattern and nuclear localization were confirmed by immunohistochemical study. Furthermore, the bacterially expressed zic protein containing the zinc finger domains bound to the GLI‐binding sequence. These findings suggest that zic is one of a number of nuclear factors involved in both differentiation in early development and maintenance of properties of the cerebellar granule cells.