, 2000 and Jacquet et al., 2009). Due to severe defects in multiple organ systems, including the lung, most foxj1 null mice die within 3 days after birth ( Brody et al., 2000). Despite a previous report ( Jacquet et al., 2009), we did not obtain any null mutants surviving past P7 in more than ten litters from crosses using the same foxj1-heterozygous mice ( Brody et al., 2000). To address nervous system-specific questions, we generated a conditional floxed allele of the foxj1 gene ( Figure 4A). We crossed our foxj1-flox (foxj1Flox/+) line to germline β-actin-cre mice to generate

a knockout allele (foxj1-KO). We then crossed this foxj1-KO (foxj1KO/+) allele to a nestin-cre driver ( Tronche et al., 1999) and foxj1Flox/Flox mice, and compared phenotypes between nestin-cre; foxj1KO/Flox (cKO) and nestin-cre; foxj1+/Flox (control)

littermates. At birth, we could not detect histological differences selleck chemicals in brain sections between control and cKO littermates, and lateral ventricle size in P3 cKO mice was comparable to controls ( Figure S5A and data not shown). The cKO mice lived without obvious signs of defect until after P7, when hydrocephalus appeared from the lack of multicilia on maturing ependymal cells ( Figure S5B). Staining of P5 cKO brain sections confirmed the removal of Foxj1 protein, normally expressed by the ependymal layer in control animals ( Figure S5C). IHC staining on brain ventricular wall whole mounts from P3 control and cKO mice showed that while Ank2 was normally expressed, Ank3 expression was absent from the developing SVZ niche in cKO mice (Figure 4B). This loss Olaparib in vivo was confirmed by western blot analyses of differentiated pRGPs, also showing concurrent reduced levels for β2-Spectrin and α-Adducin (Figure 4C). IHC staining on ventricular wall whole mounts from P6 mice

with antibodies against S100β and Glast showed that while pRGPs from control mice had matured into S100βhi/Glastlo ependymal cells, those from mutant mice remained largely S100βlo/Glasthi, resembling immature pRGPs (Figure 4D). To determine if this phenotype was due to a failure of ependymal differentiation, or the generation of additional Glast+ progenitors, we introduced by breeding the Foxj1-GFP transgenic reporter allele into the cKO background to visualize the fate of GFP+ pRGPs. Sitaxentan The possibility that Foxj1 autoregulates the 1 kb human Foxj1 promoter in the Foxj1-GFP transgene appeared low since sequence analyses showed no predicted Foxj1-binding sites (Lim et al., 1997 and Badis et al., 2009) within this promoter region (data not shown). In cKO mutant mice at P6, we detected robust Foxj1-GFP expression along the lateral ventricular surface, but these GFP+ cells continued to express Glast with little to no S100β expression (Figure 4E). These results showed that in cKO mice, the ventricular wall is populated by Foxj1-GFP+ progenitors destined to become SVZ niche cells but failed to fully differentiate into S100β+ ependymal cells.