We have also identified an intimate link between PHF6 and the PAF1 transcription elongation complex that plays an essential role

in neuronal migration in the cerebral cortex. Finally, we have identified Neuroglycan C/Chondroitin sulfate proteoglycan 5 (NGC/CSPG5) as a downstream target of PHF6 and the PAF1 complex in the control of neuronal migration. Our findings define a pathophysiologically relevant cell-intrinsic transcriptional pathway that orchestrates neuronal migration in the cerebral cortex. To interrogate PHF6 function in the mammalian brain, we first characterized the expression of PHF6 in the developing 17-AAG cerebral cortex. We found that PHF6 was highly expressed during early phases of development in primary cortical neurons and in the developing mouse brain (see Figures S1A and S1B available online). PHF6 AZD9291 concentration was broadly expressed in the mouse cerebral cortex at embryonic day 17 (E17) (Figure S1C). The temporal profile of PHF6 expression raised the possibility that PHF6 might play a role in cortical development. To determine PHF6 function in cortical development, we used a plasmid-based method of RNA interference (RNAi) to acutely knockdown PHF6 in the developing cerebral cortex (Gaudilliere et al., 2002). Expression of three short hairpin

RNAs (shRNAs) targeting distinct regions of PHF6 mRNA induced knockdown of exogenous PHF6 protein in 293T cells and endogenous PHF6 in primary mouse cortical neurons (Figures 1A, 1B, S1D, and S1E). We next employed an in utero electroporation method to induce knockdown of PHF6 in the developing mouse cerebral cortex in vivo. The PHF6 RNAi plasmids were electroporated together with a plasmid encoding GFP in the developing cortex in mice at Mephenoxalone E14, when superficial layer neurons are generated. Embryos were allowed to

develop in utero until E19, and brains were harvested and subjected to immunohistochemical analyses. We first confirmed that PHF6 RNAi triggered the downregulation of endogenous PHF6 in the cerebral cortex in vivo (Figures 1C and S1F). Upon characterizing the consequences of PHF6 knockdown on cortical development, we found a striking migration phenotype. Neurons in control animals differentiated and migrated properly to the superficial layers of the cortical plate. By contrast, cortical neurons in PHF6 knockdown animals failed to migrate to the proper location in the upper cortical plate (Figures 1D and 1E). PHF6 RNAi reduced the percentage of neurons reaching the upper cortical plate by 2- to 3-fold and increased the percentage of neurons in the intermediate zone by 3- to 5-fold. The extent of the migration defect correlated with the degree of PHF6 knockdown (Figure 1A).