Together, these results suggest that PHF6 knockdown specifically impairs the radial migration of neurons in the developing cerebral cortex. To determine whether the PHF6 RNAi-induced migration phenotype Afatinib is due to specific knockdown of PHF6, we performed a rescue experiment. Expression of rat PHF6 (PHF6-Res), which contains two mismatches with shRNA-2 targeting mouse PHF6, was refractory to PHF6 RNAi (Figure 1F). Importantly, expression of PHF6-Res in PHF6 knockdown animals largely restored the normal migration pattern of cortical neurons (Figures 1G and 1H). These data indicate that the PHF6 RNAi-induced migration phenotype is
the result of specific knockdown of PHF6. We next asked whether the PHF6 knockdown-induced migration phenotype is relevant to intellectual disability. The patient mutation C99F targets a conserved cysteine residue in PHF6’s first PHD domain (Lower et al., 2002) and impaired the MK 2206 ability of PHF6-Res to drive migration in the cerebral cortex in vivo in the background of PHF6 RNAi (Figures 1G and 1H). Likewise, deletion
of the C-terminal 86 amino acids, which also causes BFLS (Berland et al., 2011), impaired PHF6-dependent neuronal migration (Figures 1G and 1H). These data suggest that impaired neuronal migration might underlie the pathogenesis of BFLS. Having established a critical role of PHF6 in neuronal migration, we next asked how PHF6 functions in neurons at the cellular level. Radial neuronal migration in the cerebral cortex results from major cell morphological rearrangements, including the transition from multipolar to bipolar neuronal morphology in the intermediate zone and extension of the leading process toward the pia (Ayala et al., 2007; Kriegstein and Noctor, 2004; Nadarajah et al., 2001). Knockdown of PHF6 substantially increased the number of multipolar neurons and concomitantly reduced the number of bipolar neurons in the intermediate zone in the cerebral cortex in E17 embryos (Figures 2A, 2B, and 2D). The remaining bipolar neurons in the PHF6 knockdown embryos harbored
a thick leading process with Tolmetin numerous filopodia-like protrusions (Figure 2C). In other analyses, PHF6 knockdown dramatically increased the percentage of migrating neurons that lacked a leading process or that had a short, poorly developed, or aberrantly branched leading process (Figures 2E and 2F). These data suggest that PHF6 plays a critical role in the multipolar-to-bipolar transition and the morphogenesis of the leading process in migrating neurons. We next determined the mechanism by which PHF6 orchestrates neuronal migration. Immunocytochemical analyses suggested that PHF6 protein localized to the nucleoplasm in primary cortical neurons, consistent with the possibility that PHF6 might regulate transcription (Figure S1G) (Lower et al., 2002).
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