br Materials and methods br Results br

Materials and methods
Results
Discussion An interesting discovery was that mTORC1 did not affect neurite density, as measured by neurite length divided by number of neurons, in the initial period in neuronal differentiation. Considering that the number of neurons did not change over time but the neurons shrank in size in rapamycin-treated mESCs, the decreased parameter observed at 48–72h could be primarily due to the loss of neurite length. It thus can be inferred that rapamycin did not destroy the distribution of neurites, as S6K Thr389 had already significantly decreased at 24h after EB dissociation (Fig. 4). This phenomenon seems to differ from many cell types such as HEK 293T, HeLa, and MEF cells, in which cell death occurs a few hours after treatment with high concentrations of rapamycin. Similarly, the neurite network was significantly disrupted within 24–72h of treatment with wortmannin. Differences in the effects of rapamycin and wortmannin may simply be attributed to different efficacies of the drugs or dosages. Furthermore, comparing morphological and signal differences between rapamycin and wortmannin treatment, phosphorylated Akt might also play a role in neurite outgrowth. Nevertheless, raptor/mTORC1 might be a specific regulator by which neuronal differentiation may become more mTORC1-dependent after differentiation for 48–72h. The time-dependence of the importance of S6K and rapamycin in development of neurons in vivo has been reported. During differentiation of extending neural processes in mice and Drosophila, S6K was reported to play an increasingly important role in the increase in cell size (Bateman and McNeill, 2004; Shima et al., 1998; Zhang et al., 2000). Hence, we suggest that neurons derived from mESCs rely on raptor/mTORC1, and this process may be development dependent. Another interesting observation is that phosphorylation of S6K, but not 4EBP1, was decreased in rapamycin- or raptor RNAi-treated neurons derived from mESCs, and this decrease was associated with the loss of neurons/neurites. Both S6K and 4EBP are direct downstream Talabostat mesylate cost of mTORC1, but their differing functions in in vivo systems of cell differentiation have been reported. In undifferentiated photoreceptor cells of the ommatidia in Drosophila, mutation of TSC leads to a profound impairment in the timing of differentiation in these cell clusters. S6K, but not 4EB-P1, found to act downstream of TORC1 in this pathway (McNeill et al., 2008). Furthermore, Mainwaring and Kenny demonstrated a novel mechanism whereby eIF4E and S6K are differentially regulated in proliferating cerebellar neural precursors by SHH (Mainwaring and Kenney, 2011). Therefore, the observation of a difference between S6K and 4EBP1 phosphorylation in neurons either in vivo or in vitro such as in our mESC-derived neuronal model implies that additional regulation might be involved. The homozygote raptor gene trap clones verified by PCR and Southern blot analysis demonstrated that the gene is indeed being “trapped,” so that no proteins are expressed. Despite the genotype, the faint raptor band seen in the western blot, as well as subsequent immunoprecipitation for MS-SPEC analysis (data not shown), precludes us from claiming that these clones are knockouts. Since the intron-trapping vector contained a splice acceptor, it is highly possible that these clones developed a “leak” in the gene-trap approach due to a skip in splicing. Even a low level of raptor expression resulting either from the gene trap or RNAi knockdown caused a decrease in EB size, which might subsequently result in the failure of neuronal differentiation. Raptor knockout mice die early in development (Guertin et al., 2006). Nevertheless, Drosophila mutants or mice containing inactivated S6K, one of mTORC1 direct substrates, survived development and exhibited a normal body shape, but a 50% decrease overall in body size due entirely to a decrease in cell size, while the overall number of cells was unaltered (Montagne et al., 1999; Shima et al., 1998). The differences caused by raptor and its downstream effector S6K on development suggest that raptor could have a broader effect on biological function, in addition to cell growth/size.