Novel transcripts expressed in both radial glial lineages

Importantly, further novel genes were identified in the present transcriptome analysis of distinct subsets of RGCs, such as mRNAs with only a Riken code which have not even been classified so far. The expression of some of these unknown genes was also confirmed to be confined to the VZ of the cortex during development. Thus, these genes might have novel and important functions in maintaining RGCs in an undifferentiated state or inducing direct neurogenesis in the cortex during development. For example, the gene 2300002D11Rik was shown in this study to be localized in the nucleus of only a subset of RGCs in vivo and

additionally in neurons at mid-neurogenesis in the mouse cortex. This interesting expression pattern was thus consistent with the higher expression of this mRNA in only one subset of RGCs - the neurogenic set of radial glia, observed in our transcriptome analyses. Strikingly,

the mRNA and protein of the 2300002D11Rik was present specifically in stem and progenitor cells in the adult SEZ and OB - one of the adult neurogenic regions. Moreover, I found the transcription factor AP2γ higher expressed in the other lineage of RGCs – the radial glia subset largely self-renewing and giving rise to basal progenitors, as mentioned above. Interestingly, AP2γ was also expressed in the adult neurogenic regions, namely in the SEZ and DG. Thus, the expression of 2300002D11Rik and AP2γ in the progenitors of the embryonic and adult brain suggests that both lineages found in the developing cortex are maintained in the adult brain. However, these lineages are only maintained in the neurogenic regions of the adult brain but severe changes seem to happen in non-neurogenic regions, as for example the cortex.

Several other genes were found in this transcriptome analysis, whose function is still not known. Future functional analysis of these novel genes may give new insights into the molecular mechanisms regulating both progenitor lineages in the embryonic and adult mouse brain.

8.1.5 Novel insights into transcriptional changes in radial glial cells at the end of neurogenesis

At late developmental stages (E18 in the mouse) cells are switching from neurogenesis to gliogenesis (Qian et al., 2000; Temple and Qian, 1995). To elucidate the transcriptome of RGCs at the end of the neurogenic period we looked at genes differently regulated compared with isolated E14 subsets of RGCs. Consistent with the decreased neurogenesis at this developmental stage, transcription factors related to a neurogenic fate (e.g. Pax6, Neurog1/2, Emx1) were down-regulated similar to genes involved in Wnt and BMP signalling pathways [(see e.g. (Chenn and Walsh, 2002)]. These data suggest that Wnts and BMPs may promote self-renewal or maintenance of undifferentiated radial glia. Indeed, Wnt signalling appears to regulate proliferation and differentiation of neuronal lineages in a stage-specific and cellular context-dependent manner (Chenn and Walsh, 2002; Hirabayashi et al., 2004). BMPs promote astroglia differentiation during embryogenesis and in dorsal regions of the telencephalon they not only promote astrocyte fate, but also inhibit neurogenesis and oligodendrocytogenesis (Mabie et al., 1997; Mehler et al., 2000; Nakashima et al., 2001). Some members of the BMP family, such as BMP2 and BMP4, have been shown to have anti- neurogenic effects in several systems, where they inhibit proliferation and/or induce apoptosis of neural progenitor cells during development (Furuta et al., 1997; Mabie et al., 1999; Shou et al., 1999). In some systems, however, BMP2 and BMP4 have been reported to have positive effects on neurogenesis, for example by promoting neuronal differentiation (Li et al., 1998). BMP6 and BMP7, members of a separate BMP subfamily, have been reported to have both positive (Arkell and Beddington, 1997; Furuta et al., 1997) and negative (Li et al., 1998; Shou et al., 1999) effects on neurogenesis. Interestingly, Wnt signalling indirectly regulates gliogenesis by inducing BMPs in neuronal cells (Kasai et al., 2005). Thus, cooperation between Wnt and BMP signalling seems to play an important role in determining the sequence of neurogenesis and gliogenesis during development and their tight regulation may be important to terminate neurogenesis in the cortex.

mRNAs encoding for proteins positively regulating cell cycle progression also decreased in E18 radial glia, consistent with a decreased proliferation rate and a slower cell-cycle of RGCs at late developmental stages (Caviness et al., 2003). This finding is in agreement with the gene expression profiling of isolated progenitors Lex+ cells at E17 where genes maintaining proliferation were reduced (e.g. eif3s7, cdk9 and sin3a also found in our arrays) (Abramova et al., 2005).

Interesting pathways that were up-regulated at this stage included Notch signalling (e.g. Notch2 and Dner), genes involved in gliogenesis (FABP7, TN-C and Erbb4), oligodendrogenesis (Olig1/2 and Pdgfα) and in the oligodendrocyte lineage (Brn-1 and Brn- 2) (Schreiber et al., 1997). Indeed, a third wave of Emx1-derived oligodendrocytes progenitors was described in the cortex around birth, demonstrating that these oligodendrocytes were generated from endogenous cortical progenitors (Kessaris et al., 2006). As our data showed that oligodendrocytes related genes start to be up-regulated in RGCs at late developmental stages, this suggests that these RGCs might generate the oligodendrocytes in the early postnatal cortex. In addition, other reports show that VZ cells in the motoneuron progenitor domain of the ventral spinal cord also express oligodendrocyte markers (Olig2) (Mukouyama et al., 2006). The isolation of these Olig2+ progenitors show that they loose their neurogenic potential in the gliogenic period and therefore they are not multipotent stem cells (Mukouyama et al., 2006). Early Olig2 positive cells (from murine E9.5) generate neurons and oligodendrocytes, while late Olig2 positive cells generate only oligodendrocytes upon transplantation (Mukouyama et al., 2006). Thus, throughout the CNS, oligodendrocytes appear to be the late progeny of radial glia, accordingly with our transcriptome analysis.

Another interesting finding related to the transcriptome analysis of radial glia at the end of neurogenesis is related to the down-regulation of chromatin remodeling genes, such as Chrac1 and Smarca2/a4/a5/b1/c1. These novel results may indicate that the down-regulation of these factors is important to inactivate certain genes involved in self-renewal of RGCs and for closing their neurogenic potential. Indeed, nucleosome remodeling factors were previously shown to be important to increase the accessibility of chromatin and thus activating gene expression (Baker et al., 2003; Sif et al., 1998; Simone, 2006). Furthermore, recent reports show the important role of Smarca4 in the maintenance of proliferating neural progenitors (Matsumoto et al., 2006).

We compared the transcriptome of pure RGCs isolated at the end of neurogenesis with the subsets of RGCs at E14 and the ES cells-derived radial glia using different culturing conditions (Bibel et al., 2004; Conti et al., 2005) by divisive clustering analysis. Interestingly, E18 isolated RGCs had a very distinct transcriptome comparing to ES-derived RGCs and were also very distinct from both subtypes of radial glia sorted from E14. Thus, E18 RGCs showed very pronounced changes at the transcriptome level.

In summary, our results evidence the fact that we were able to isolate two different lineages of RGCs in the cortex at mid-neurogenesis and we further identified the molecular

differences between these distinct sets of radial glia. One set consisted of radial glia that expressed canonical Pax6 and directly generated neurons, whereas the other set of radial glia expressed endogenous low levels of canonical Pax6 and had less neurogenic capacity. This second population generated only few neurons indirectly through the generation of basal progenitors which were enriched in this fraction. Importantly, both subsets of RGCs were very distinct from non-neurogenic RGCs isolated at the end of neurogenesis at the transcriptome level. Thus, our transcriptome analysis gave new insights into the potential fate determinants of distinct lineages of RGCs in the developing mouse cerebral cortex. The functional relevance of the two lineages isolated at mid-neurogenesis was confirmed by our functional analysis of the AP2γ transcription factor.

8.2 Expression and function of the transcription factor AP2γ in a subset of