INFORMACIÓN SECUNDARIA

Notably, expression of Dlx2 in the adult subependymal zone had been reported previously (M. H. Porteus et al., 1994; F. Doetsch et al., 2002). However, little is known about the expression profile of Dlx2-positive cells in the subependymal zone and about their fate in the olfactory bulb. I therefore investigated the function of Dlx2

2.9 Candidate genes 35

in the adult subependymal zone and in neuronal subtype specification for olfactory interneurons.

2.9.1 The role of Dlx2 in adult neurogenesis in regard to Pax6

Dlx1 & Dlx2 belong to the key molecular regulators for GABAergic neuron specification during development besides Gsh1 & Gsh2 since the respective double mutant mice loose virtually all GABAergic telencephalic interneurons prior to birth (S. A. Anderson et al., 1997a; D. D. Eisenstat et al., 1999; H. Toresson et al., 2000; H. Toresson and K. Campbell, 2001). However, the function of these transcription factors in adult neurogenesis is yet unknown as most of the mouse mutants die perinatally. We therefore aimed to determine here whether Dlx transcription factors maintain their region-specific expression in the adult forebrain and whether they still act to determine all or only a specific subset of olfactory interneurons. Expression of Dlx2 had been observed in transit-amplifying progenitors and neuroblasts (M. H. Porteus et al., 1994; F. Doetsch et al., 2002). Furthermore, Pax6 had been shown to be present in virtually all neuroblasts and Olig2 as a marker for transit-amplifying progenitors (M. A. Hack et al., 2005). Given this co-existence in the same cell types I examined whether Pax6 is required for Dlx2 function and if these transcription factor would interact on the molecular level.

Fig. 15: Schematic summarizing the known network of some transcription factors in the adult subependymal zone.

The transcription factors Dlx2 and Olig2 are both present in transit-amplifying progenitors (TAPs), whereas Pax6 is expressed in neuroblasts. The aim of the study was to study the function of the transcription factor Dlx2 in regard to Pax6.

2.9.2 Tbr1 & Tbr2

Since previous reports demonstrated the contribution of the Emx1-derived, Pax6 expressing dorsal telencephalon to the adult subependymal zone (K. M. Young et al., 2007; S. Willaime-Morawek and D. van der Kooy, 2008), I investigated the regionalization of the subependymal zone in regard to the presence of transcription factors of the embryonic dorsal telencephalon. Furthermore, I examined if the dorsal

wall would generate glutamatergic neurons destined for the olfactory bulb. Consequently this would suggest that neural stem cells of the dorsal subependymal zone recapitulate their developmental program. In the adult subependymal zone Pax6 is expressed in a gradient from dorsal to ventral, whereas Dlx transcription factor are exclusively expressed in the lateral ventricular wall. During embryonic development, dorsal progenitors generate predominantly glutamatergic neurons, in a Pax6-dependent manner, including those of the olfactory bulb (T. Nomura and N. Osumi, 2004; T. T. Kroll and D. D. O'Leary, 2005; V. Nikoletopoulou et al., 2007). Thus, the entire population of olfactory projection neurons is thought to derive from a Pax6-expressing territory and Pax6 is crucial for the formation of the olfactory bulb (T. L. Dellovade et al., 1998; D. Jimenez et al., 2000). In the developing cerebral cortex, Pax6 regulates Neurogenin1 and Neurogenin2 which are crucial to specify at least deep cortical layer neurons towards a glutamatergic fate (C. Schuurmans et al., 2004). In addition, in the cerebral cortex and hippocampus Pax6 and its target Neurogenin2 regulate expression of the T-box transcription factors Tbr1 and Tbr2 expressed in early postmitotic glutamatergic neurons and their intermediate progenitors (R. F. Hevner et al., 2006). Given the glutamatergic progeny of the dorsal telencephalon during development and its contribution to the adult subependymal zone, I searched for progenitor cells in the dorsal part of the subependymal zone that may generate glutamatergic neurons in the adult olfactory bulb.

Fig. 16: Schematic depicting the dorsal region (dSEZ) and lateral region (latSEZ) of the adult subependymal zone.

Previous study demonstrated the regionalization of the adult subependymal zone. However, during development glutamatergic neurons are generated in the dorsal telencephalon forming later the dorsal wall of the subependymal zone (dSEZ). Therefore, we investigated if transcription factors would be expressed in the dSEZ that governs the generation of glutamatergic olfactory neurons. OB = olfactory bulb; RMS = rostral migratory stream; dSEZ = dorsal subependymalzone; latSEZ = lateral subependymal zone, vSEZ = ventral subependymal zone.

dSEZ _{latSEZ or vSEZ}

OB RMS

3 Aims of this study 37

3

Aims of this study

The postulated regionalization of the adult subependymal zone and the transcriptional network specifying olfactory interneurons is still debated and should be addressed in this study.

In my thesis, the regionalization of the adult subependymal zone should be clarified. The candidate gene Dlx2 should be studied in regard to regionalization, expression pattern and function in adult neurogenesis and subtype specification in the olfactory bulb. The function of Dlx2 should be examined in vivo by the construction and injection of retroviral vectors for gain- and loss-off-function experiments. Furthermore, the in vivo results should be supported by in vitro models (neurosphere assay and direct plated subependymal zone progenitors) and in vivo time-lapse imaging should be employed to further elucidate the function of Dlx2.

In addition, the contribution of the dorsal subependymal zone which is derived from the embryonic dorsal telenecpehalon should be elucidated in regard to glutamatergic olfactory neurogenesis. Transcription factors involved in cortical glutamatergic neurogenesis should be identified in the dorsal adult subependymal zone. If these factors would be present in these regions their progeny in the rostral migratory stream and in the olfactory bulb should examined using labelling methods like the DNA base analogue BrdU, retroviral injections, as well as transgenic mouse lines

4

Results

4.1

A Dlx2- and Pax6-dependent transcriptional code for