We then generated lentiviral construct-expressing shRNA against mouse Ank3, and tested this in NIH 3T3 cells, which knocked down greater than 95% of endogenous Ank3 after lentiviral infection (Figure S4A). We next made lentivirus coexpressing this shRNA and GFP under control of the

1 kb human Foxj1 promoter (the same promoter used to generate the Foxj1-GFP transgene) (Ostrowski et al., 2003), and infected pRGP cultures 24 hr after plating. Lentiviral infection of pRGPs was highly efficient as more than 90% of multiciliated cells (assessed by γ-tubulin/DAPI staining) selleck chemicals became GFP+ after differentiation (Figure 3C and data not shown). While control virus-infected pRGPs upregulated Ank3 in clusters as normal, we were able to knockdown this expression with the Ank3 shRNA virus (Figure 3C). As GFP expression in infected cells did not become bright enough for live imaging until 3–4 days after infection (too late for following cellular clustering in real time), we used antibody staining to quantify the ability of infected pRGPs to cluster after differentiation (Figure S4B). Counting cells

stained with GFAP, γ-tubulin, and Phalloidin, we found that Ank3 shRNA-infected pRGPs had significantly reduced numbers of clustered structures when compared to control virus-infected cultures (Figure 3D). To confirm these findings in vivo, we performed stereotactic injection of control Selleck HSP inhibitor and Ank3 shRNA lentiviruses into P0 mice, specifically targeting pRGPs through striatal injections (Merkle et al., 2004). Ventricular whole-mount staining 5 days after lentiviral injection showed that control pRGPs were able to assemble into clustered structures, with Ank3+ ependymal cells exhibiting large apical surface areas surrounding Ank3− cells with small apical surfaces (Figure S4C). In contrast, Ank3 knocked-down pRGPs failed to organize into clusters along the ventricular surface, and retained a smaller apical surface area (by Phalloidin staining) as compared to neighboring

cells with intact Ank3 expression (Figure S4C). Furthermore, whereas the Ank3+ pRGPs Farnesyltransferase had largely downregulated immature ependymal marker Glast (Figure 3E), Ank3 knocked-down pRGPs retained high-level Glast expression, showed disorganized patterning, and failed to differentiate into mature multiciliated ependymal cells (Figure 3E). Since striatal lentiviral injection can only target a small number of pRGPs, we would like to remove Ank3 function in vivo. One strategy is to delete its upstream regulator in pRGPs to prevent Ank3 expression. To our knowledge, transcriptional regulation of ank3 (or any of the other Ankyrins) is not known. One candidate for such control, since its expression appears before Ank3 in pRGPs ( Figure 1), is the transcription factor Foxj1. It is a well-established regulator of motile-cilia formation ( Yu et al.