The results of the zMsi1 expression analysis (Fig. 5 and Fig. 6) showed that zMsi1 was expressed in the CNS, including the telencephalon, and could be involved in neurogenesis in this region. Zebrafish miR-9 (z-miR-9) is expressed in the telencephalon from 20 to 24 hpf, and inhibits the in vivo expression of Her5 and Her9 mRNAs, mouse Hes basic Ponatinib purchase helixloop-helix transcription factor orthologs, and neural repressors

( Leucht et al., 2008). Interestingly, mMsi1 regulates Hes expression by binding to the 3′UTR of the m-numb mRNA and controlling its translation ( Imai et al., 2001). Alternatively, Msi1 enhanced the expression of the miR-9 directed reporter conjugated to the Nr2e1 3′UTR ( Shibata et al., 2011). Our recent study reported that Msi1 regulates miRNA processing of the let-7 family member mir-98, which acts as a Lin28-enhancer protein during early neural differentiation of ES cells ( Kawahara et al., 2011). These results suggest that zMsi1 also may be involved in neurogenesis and tumorigenesis via miRNA processing and translational control of its direct targets. However, it will be essential to identify bona fide RNA target genes of Msi in a genome-wide screen to predict candidate downstream effectors in development. Then, the involvement of zMsi regulatory

pathways in neural development could be clarified by in vivo manipulations http://www.selleckchem.com/products/bmn-673.html using our zebrafish model. Further analysis of Msi function using this novel zebrafish model will provide new insights into human neurological diseases that are linked to a failure in normal brain development. For this study, the RIKEN WT (RW) zebrafish was used as the control wild-type strain of D. rerio. The HuC:GFP (Tg(elavl3:EGFP)zf8) transgenic D. rerio ( Park et al., 2000) expressing GFP as a neural tissue marker was obtained from the RIKEN bioresource center. The completely transparent ifoxetine samples shown in Fig. 5 and Fig. 7 were prepared by treating fertilized eggs with 0.1 mM phenylthiourea (PTU) to block pigment formation. All experimental procedures

were approved by the Institutional Recombinant DNA Committee, and the Animal Care and Use Committee of Keio University. Total RNA from different stages of zebrafish fertilized eggs and embryos and from adult brain were isolated using the Trizol reagent (Invitrogen) according to the manufacturer’s instructions. First strand cDNA synthesis was performed with 1 μg total RNA and oligo-d(T)12–18 primers at 42 °C for 50 min according to the manufacturer’s instructions (Superscript III, Invitrogen). Cloning of zMsi1 was performed using Taq DNA polymerase (Invitrogen) PCR with the following primer sets: zMsi1F, 5′-CTTTTCTCGCACCAGACTCGG-3′, and zMsi1R, 5′-TCAGAGAGGGTCCAGCTTCAA-3′; zMsi1F_XbaI, 5′-AATCTAGAATGGAATCGGAAGGCAGCCA-3′, and zMsi1R_XbaI, 5′-AATCTAGAATGGTAGCCATTAGTAAATG-3′, in the pGEM-T-Easy cloning vector (Promega).