These pRGPs also progressed from Glasthi/S100βlo to Glastlo/S100βhi during differentiation ( Figure 2A), consistent with our previous observation for postnatal ependymal differentiation in vivo ( Kuo et al., 2006). Quantifying γ-tubulin cluster staining as

a percentage of DAPI nuclear staining, 64% ± 6.5% standard deviation (SD) of total cells after completion TSA HDAC of differentiation became multiciliated. We noticed that differentiated ependymal cells often clustered in culture, and scanning electron microscopy analyses showed multiciliated cells arranged in clusters around monociliated cells (Figure 2B and Figure S2A), much like their in vivo organization (Mirzadeh et al., 2008). The monociliated cells within ependymal clusters were also GFAP+ in culture (Figure 2C and Figure S2B). To understand the processes leading

to pRGP clustering in vitro, we performed live cell imaging using the Foxj1-GFP reporter mouse line (Ostrowski et al., 2003). We observed that shortly after plating, MLN0128 mouse there was an increase in the number of GFP+ pRGPs, followed by upregulation of GFP expression and cellular clustering (Movie S2). Expansion in the number of GFP+ cells was accomplished by both progenitors starting to express GFP as well as by cell division (Movie S3). It is interesting to note that the majority of GFP+ pRGP clustering took place 3–4 days after plating, prior to the appearance of multicilia, which began 7–8 days after plating (Movie S2 and Figure 2D). Once the clustering was complete, these structures were positionally stable (Movie S2). IHC staining revealed that the clusters represented multiciliated Foxj1-GFP+ arranged around monociliated GFP− cells (Figure S2C). To understand if this pRGP culture may be useful for tackling Ank3 function, we saw that during in vitro differentiation, pRGP clusters upregulated Ank3 along their cell borders, prior to multicilia formation (Figure 3A). Western blot analyses of pRGP cultures confirmed robust increase

in the 190 kDa Ank3 protein (known to be an epithelial-specific splice form) (Kizhatil et al., 2007) after differentiation, as well as Ank3-associated proteins β2-Spectrin and α-Adducin (Figure 3B). We wanted to know whether Ank3 expression/localization is dependent on multicilia Rolziracetam formation in SVZ ependymal cells. Using a tamoxifen-inducible foxj1-CreERt2 transgene ( Rawlins et al., 2007), we deleted Kif3a, a critical molecular motor for cilia formation ( Marszalek et al., 2000), from postnatal pRGPs. Lineage-tracing analyses of the foxj1-creERt2; rosa26-YFP reporter mice injected with tamoxifen at birth and analyzed at P14 showed that Foxj1-CreERt2 can target multiciliated ependymal cells ( Figure S3A). We generated clonal deletion of Kif3a in pRGPs by low-dose tamoxifen injection into newborn foxj1-creERt2; kif3aKO/Flox mice.