A zMsi1 antisense probe detected endogenous mRNA in some parts of the brain, spinal cord and eyes ( Figs. 5A, C). The 48-hpf sample with antisense probe signaling pathway showed restricted expression of zMsi1 in the forebrain in embryos and

no signal was detected in the spinal cord ( Fig. 5C). These results indicate that zMsi1 is expressed mainly in the CNS in zebrafish embryos and we hypothesized that zMsi1 may play important roles in the development of the CNS in vivo. To evaluate Msi1 protein levels at each developmental stage, we performed immunoblotting with a rat monoclonal anti-mouse Msi1 antibody (14H1) that was described previously (Kaneko et al., 2000). Protein lysates were prepared from whole embryos or isolated heads of wild-type zebrafish, homogenized at 2, 3, 4 days and 5 months post-fertilization. The same amount of mouse brain lysate from embryonic day 14.5 was loaded as a positive control (Fig. 6A). The amounts of protein loaded in each lane were referenced with that of

internal control antibodies against α-Tubulin and β-Actin (Fig. 6B). The recognition sequence for the 14H1 monoclonal antibody is highly SNS-032 research buy conserved (nine out of ten amino acids are identical between mouse and zebrafish) (Fig. 1A, blue bar). A single band at approximately the size of mouse Msi1 (362 amino acids) was detected at each stage. Mouse Msi1 is only 13 amino acids longer than zMsi1L (349 amino acids). Several faint non-specifically stained bands were present in the zebrafish samples. The zMsi1 protein was detected at day 2 (48 hpf), and Ketotifen the expression levels gradually increased in an age-dependent manner in the 2–7-day-old zebrafish (Fig. 6A). In adult zebrafish with an age of 5 months, zMsi1 expression was much higher than in embryonic stages. By contrast, in the mouse, the level of Msi1 is highest in the early embryonic period, and its levels are much lower in the adult brain. Taken together, these results show that the expression profile of Msi1 in zebrafish was different from that in mouse. Protein localization of zMsi1 was evaluated via immunohistochemistry

in day 2 (48 hpf) zebrafish embryos (Figs. 6C–G). Many cells in the CNS and the eyes expressed zMsi1, which exhibited an expression pattern similar to that of the proliferative cell marker, PCNA (Fig. 6C). Cells positive for cytoplasmic Msi1 were co-labeled with anti-PCNA in the forebrain, midbrain, hindbrain and eyes (Figs. 6D–G). To evaluate the functions of Msi1 in zebrafish development, we constructed and injected MOs into one-cell stage wild-type zebrafish embryos to knock down expression of zMsi1. To evaluate the survival rate and observe specific phenotypes in the MO-injected group, the following controls were prepared: non-injected (injection minus) wild type ( Fig. 7A) and randomized sequence MO-injected wild type ( Fig. 7B).