Regionalization and fate specification in neurospheres: the role of Olig2 and Pax6

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Abstract

Neurosphere cultures are widely used to propagate multipotent CNS precursors, but their differentiation into neurons or oligodendrocytes is rather poor. To elucidate fate determination in this system, we examined the expression and function of candidate transcription factors in neurospheres derived from different CNS regions during development and adulthood. We observed prominent down-regulation of most transcription factors present in telencephalic precursors upon growth factor exposure in neurosphere cultures while Olig1 and Olig2 expression was strongly up-regulated. Interference with Olig2 in neurospheres revealed its role in self-renewal during expansion and for the generation of neurons and oligodendrocytes during differentiation. We further show that neurogenesis becomes fully Pax6-dependent in the neurosphere culture system, independent of the region of origin, and that Pax6 overexpression is sufficient to direct almost all neurosphere-derived cells towards neurogenesis. Thus, a pathway combining transcription factors of dorsal and ventral regions is activated in the neurosphere culture model.

Introduction

The isolation of multipotent cells from the adult CNS Kilpatrick and Bartlett, 1995, Reynolds and Weiss, 1996 has prompted great hopes for reconstitution of degenerating neurons or oligodendrocytes in neuropathological conditions. However, precursors isolated from the adult telencephalon and propagated as neurospheres generate disappointingly few neurons, both in transplantation paradigms as well as in differentiating conditions in vitro Fricker et al., 1999, Herrera et al., 1999, Song et al., 2002, Winkler et al., 1998. This is even more surprising since adult neural precursors in vivo generate neurons life-long, suggesting that the in vitro expansion model somehow interferes with the normal cascade of specification.

Adult neurogenesis is currently thought to progress by a sequential manner of fate restriction deduced from cell lineage analysis. A slow dividing precursor with astroglial features, called type B cell, generates fast-dividing transit-amplifying precursors, the type C cells Doetsch et al., 1999, Doetsch et al., 2002. Indirect evidence suggests that these precursor types are not yet irreversibly restricted to a certain lineage since they can still give rise to several cell types upon isolation in the neurosphere culture conditions Capela and Temple, 2002, Doetsch et al., 2002, Imura et al., 2003, Morshead et al., 2003 and they can alter their progeny in response to certain lesion paradigms in vivo Arvidsson et al., 2002, Decker et al., 2002. In contrast, the progeny of type C cells, the type A cells, express neuronal characteristics and seem to be specified to the neuronal fate Doetsch et al., 1999, Menezes et al., 1995. However, little is known about the molecular determinants of this sequential fate restriction. Dlx2 expression has been detected in transit-amplifying cells (Doetsch et al., 2002), reminiscent of transcription factors specific for the ventral telencephalon during development, but its role for fate specification has so far only been examined in the developing telencephalon Anderson et al., 1997a, Anderson et al., 1997b.

Therefore, it might help to look at the mechanisms involved in fate specification during development where a similar cascade of fate restriction was observed in the telencephalon. Cell lineage analysis using replication-incompetent retroviral vectors revealed that most telencephalic precursors generate only a single cell type during neurogenesis, although some precursors also give rise to large and widespread clones containing multiple cell types Grove et al., 1993, Luskin et al., 1988, Luskin et al., 1993, Price and Thurlow, 1988, Reid and Walsh, 2002, Walsh and Cepko, 1993. The intrinsic lineage bias of telencephalic precursors was revealed when single cells were isolated in vitro and also found to generate exclusively a single cell type (Qian et al., 1997). In further support of progressive fate restriction, the addition of growth factors can no longer affect the progeny of precursors isolated during neurogenesis from the cerebral cortex, but these factors still influence fate decisions of neuroepithelial cells isolated at earlier stages Götz et al., 2002, Williams and Price, 1995, Williams et al., 1991. Taken together, these data suggest that fate specification in the developing and adult CNS occurs by progressive fate restriction from multipotent towards further specified precursors.

In the developing CNS, some intrinsic fate determinants have been identified. For example, we have recently characterized the transcription factor Pax6 as a potent neurogenic determinant in precursors of the dorsal telencephalon, the anlage of the cerebral cortex Götz et al., 1998, Heins et al., 2002. Pax6 is required for most of the neurogenesis in the cerebral cortex and is even sufficient to direct postnatal astrocytes from this region towards neurogenesis. Similarly, members of the bHLH transcription factor family also play important roles in the specification of neurons and oligodendrocytes, but they also act in a tight region-specific and cell-type-specific context (Bertrand et al., 2002). These results highlight the importance of patterning in CNS development and the close link between patterning and fate determination Bertrand et al., 2002, Campbell and Götz, 2002 and raise the question about these mechanisms in neurosphere cultures. To which extent is the specification of precursors altered by the neurosphere culture system and to which extent is patterning information maintained? Indeed, region-specific differences among neurosphere cultures originating from different regions have been reported Hitoshi et al., 2002a, Hitoshi et al., 2002b, Ostenfeld et al., 2002, Parmar et al., 2002, but others found significant changes in region-specific features after expansion in neurosphere cultures Gabay et al., 2003, Santa-Olalla et al., 2003. Moreover, little is known about the differences or similarities of neurospheres derived from the adult subventricular/subependymal zone to those from different embryonic regions.

To gain some insights into the molecular mechanisms of fate specification in neurosphere cultures, we first examined the expression of candidate fate determinants in neurospheres derived from different regions of the developing CNS and the adult SVZ before and after differentiation. These results showed a pan-regional down-regulation of most transcription factors in the expansion phase of neurospheres with the exception of the Olig genes. Interestingly, a similar down-regulation of most region-specific transcription factors except the Olig genes was also observed when adherent cells were exposed to EGF or FGF2. When neurosphere cells were exposed to differentiation conditions, only Pax6 was up-regulated in numbers consistent with a role in neurogenesis, independent of the region of origin. We then evaluated the role of Olig2 and Pax6 in loss-of-function and gain-of-function experiments in neurosphere cultures.

Section snippets

Transcription factor analysis in neurosphere cultures in expansion conditions

Cells from the adult subependymal or subventricular zone (SEZ/SVZ), E14 cortex and ganglionic eminence (GE) were cultured at clonal density (Morshead et al., 2003) for four passages as neurospheres (Fig. 1) and then plated in differentiating conditions (see Experimental methods for details). Consistent with previous data, the progeny of neurospheres included few neurons (adult NS: 12 ± 5%, n = 75; E14 CTX: 9 ± 5%, n = 199; E14 GE: 9 ± 4%, n = 140) and oligodendrocytes (adult NS: 3 ± 1%, n = 45;

Discussion

Here, we described a candidate gene expression and functional analysis of intrinsic fate determinants. We found a dramatic change of region-specific differences in gene expression in cells cultured as neurospheres that retained high levels of Olig1/2 and initiated a Pax6-dependent mechanism for neurogenesis upon differentiation. The functional analysis revealed that Olig2 plays a role for self-renewal in expansion conditions of neurosphere cultures and promotes the generation of neurons and

Animals and cell culture

Here, we used C57/Bl6 and the Pax6-mutant allele Small-eye (Sey) on a DBA/C57Bl6 hybrid background Heins et al., 2002, Hill et al., 1991. Embryos were collected from time-mated mice (mice: day of plug = E0). The cortex or ganglionic eminence (GE) of C57/Bl6 mice or Sey/Sey and wildtype littermates at embryonic day (E) 14 was dissected and mechanically dissociated. The cells were spun down and 10 cells/μl were cultured in a T75 flask in 15 ml serum-free DMEM/Nut.Mix.F-12 medium (GIBCO)

Acknowledgements

We are particularly grateful to David Anderson, Kenneth Campbell and Robert Hevner for providing antibodies; to Romy Müller and Mücella Öcalan for excellent technical assistence; to Monika Falkenberger for help with histology and to Anja Brauer for excellent secretarial help. We would also like to thank Yves Barde, Benedikt Berninger, Francois Guillemot and Derek van der Kooy for discussions and helpful suggestions on the manuscript. The antibody directed against nestin was provided by the

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