We next tested the relationship between Ank3 and neuroblast production in our pRGP niche culture assay. Although no exogenous growth factors (EGF and bFGF, required for SVZ NSC renewal ex vivo) were buy Pfizer Licensed Compound Library added at any time to these primary cultures, we reasoned that perhaps the presence of Ank3+ ependymal niche cells may support NSCs and allow them to make neuroblasts during differentiation. IHC staining of pRGP culture in differentiation media 5 days after plating showed large numbers of DCX+ neuroblast clusters, with most in close proximity to Ank3+ niche clusters (arrows, Figure 8D). To determine if Ank3 expression by these niche clusters was required for neuroblast production, we used the same

shRNA strategy to efficiently remove Ank3 protein expression from differentiating pRGPs (Figure 3C and Figure S4B). This resulted in a dramatic reduction of DCX+ neuroblast clusters seen in Ank3 shRNA-treated versus control virus-treated cultures (Figure 8E). Harvesting the Ank3 shRNA-treated pRGP cultures earlier or

later during differentiation also did not show formation of DCX+ neuroblast clusters (data not shown), revealing that the defects in neuroblast production were not due to DCX+ cells dying or a delay in differentiation program. These results are in support of our in vivo observations that postnatal Ank3-mediated SVZ ependymal niche organization is required for the continued production of new neurons. To study the functional significance of SVZ niche on new neuron production, we first showed that pRGPs have an intrinsic ability to cluster into

FRAX597 molecular weight structures of the adult SVZ neurogenic niche. We discovered that the lateral membrane adaptor protein Ank3 is specifically upregulated in pRGPs destined to become SVZ niche cells, but not in stem cells, and that this Foxj1-regulated expression is necessary for pRGP assembly into mature SVZ structures. Disruption of this Foxj1-Ank3 pathway in vivo specifically removed SVZ architecture, allowing us to Ergoloid demonstrate, to our knowledge, for the first time that the mature ependymal niche is required to maintain continued production of new neurons in the postnatal brain. Our results showing that Ank3 functions in pRGPs destined to become SVZ ependymal cells, but not in future stem cells, revealed selective Ankyrin usage by a subpopulation of progenitors to establish brain ventricular wall organization. This previously, to our knowledge, undescribed function for Ankyrin is exciting both for SVZ neurogenesis and Ankyrin biology. The ankyrin gene family was first discovered over 30 years ago, but until this study, to our knowledge, no transcription factor had been linked to these proteins. While we showed here that pRGPs upregulate the 190 kDa isoforms of Ank3, mature neurons express the larger 480 and 270 kDa Ank3 isoforms ( Kordeli et al., 1995).