, 2009). Given that these adhesive contacts must be shed upon differentiation, we next investigated whether the FK228 pro-differentiation actions of Foxp4 might involve changes in N-cadherin expression or subcellular distribution. In transverse sections of the spinal cord, we noticed that there was a slight thinning of apical N-cadherin staining around the region of the pMN (Figure 3A, bracket). This difference was more clearly revealed by imaging the apical surface of the neuroepithelium in an open book preparation, which showed distinct bands of N-cadherin staining corresponding to the different

progenitor domains along the dorsoventral axis (Figures 3B–3E and S5A–S5J). N-cadherin was strikingly reduced wherever Foxp4 was present (Figures 3B and 3D–3F; averaged correlation R2 = −0.722). This antithetical pattern was specific to Foxp4 and N-cadherin as there was no correlation between the expression of Foxp4 and other AJ

components such as aPKCζ or the NPC marker Sox2 ( Figures 3B, 3C, and S5B–S5L). Under conditions of Foxp4 misexpression, the electroporated spinal cords displayed a dramatic loss of N-cadherin protein and disruption in the ultrastructure of the neuroepithelium (Figures 3G, 3K, 3M, and 3Q). These changes coincided with an aberrant distribution or loss of other AJ components Selleckchem BKM120 including β-catenin, f-actin, aPKCζ, and Par3 (Figures 3H, 3I, 3N, and 3O, and data not shown) and cytoplasmic Rolziracetam accumulation of Numb (Figures 3L, 3M, and 3R). The radial morphology of NPCs was also severely disrupted (Figures 3H, 3I, 3N, and 3O), and markers of dividing cells such as BrdU incorporation and phosphohistone H3 staining were reduced (data not shown). Nonetheless, integrin-laminin interactions at the basolateral membrane remained intact (Figures 3J and 3P), suggesting

that the effects of Foxp4 misexpression are primarily directed to apical attachments. Identical results were seen with misexpression of Foxp2 and Foxp1 (Figure S3), indicating that all of the Foxp proteins have the capacity to repress N-cadherin expression and disrupt AJs under these conditions. The combined knockdown of Foxp2 and Foxp4, in contrast, led to an ∼1.5–2-fold upregulation of N-cadherin mRNA and protein within the pMN and extensive accumulation of Numb at the apical membrane of these cells (Figures 3S, 3T, and S5M–S5Q). The effects of the shRNA constructs were specific, as the knockdown phenotype was completely reversed by coelectroporation of a Foxp4 expression vector, often resulting in the Foxp4 misexpression phenotype (Figures S2J–S2R). Together, these data indicate that Foxp2 and Foxp4 play a crucial role suppressing the expression of N-cadherin and disassembling neuroepithelial AJs (Figure 3U).