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.