The results presented here demonstrate an involvement of Shh signaling in the regulation of SVZ stem cells, leading to sustained neurogenesis, in the postnatal and adult mouse brain. Taken together, the gene expression analyses and the in vitro and in vivo experiments indicate that Shh signaling is critical for the modulation of the number of cells with stem cell properties, for the proliferation of early precursors and consequently for the production of new neurons.
Our in vivo single cell analyses indicate that Shh acts directly on GFAP+
periventricular astrocytes (B cells) and more abundant GFAP−
early precursors (C cells). In vitro results are consistent with this conclusion, although responsiveness to Shh, present in stem (B) cells, could also be induced in precursors in vitro. Indeed, transit amplifying precursors (C cells) can give rise to neurospheres in vitro under the influence of EGF, which may induce them to display stem cell properties (Doetsch et al., 2002
). Ependymal (E) cells, which were also proposed to behave as stem cells (Johansson et al., 1999
) (see Doetsch et al., 1999
; Capela and Temple, 2002
), and migrating neuroblasts (A cells) expressed Gli2
but not Gli1
, suggesting that these cells do not show the canonical response to Shh if they respond at all. Consistently, A cells did not increase their proliferation in vitro in response to Shh.
In the developing cerebral cortex, Shh acts in cooperation with EGF but Shh on its own is not sufficient to alter neurosphere size or number (Palma and Ruiz i Altaba, 2004
). We show here that Shh similarly has a proliferative effect on SVZ neurospheres in cooperation with low doses of EGF, demonstrating the conserved action of Shh as a mitogen that synergizes with EGF. The selective expression of EGFR in C cells (Doetsch et al., 2002
), and thus the ability to respond to EGF, provides additional evidence that Shh and EGF synergize in the proliferation of these early precursors. Moreover, the inhibition of adult cell proliferation and neurosphere formation by cyclopamine further proves the requirement of Shh signaling in adult stem cell lineages.
In contrast to our results in the SVZ, Shh is sufficient to induce proliferation of hippocampal precursors (Lai et al., 1993). Such a difference could suggest the endogenous production of cofactors, such as EGF, by hippocampal cells at sufficient quantities in vitro. In addition here, and in contrast to other data (Machold et al., 2003
), we show that Shh is sufficient to increase the number of neurospheres derived from SVZ cultures grown over quiescent astrocytes, indicating that in this case, such cultures may also produce sufficient levels of EGF or other cofactors. This difference might relate to the method used: it is possible that the astrocytes in the feeder layer produce enough cofactors but at low enough levels for Shh to act, whereas saturating levels of EGF mask the effects of Shh (Palma and Ruiz i Altaba, 2004
Interestingly, our present data show that Shh and EGF synergize maximally within a narrow concentration range, in a manner similar to that we described in the embryonic neocortex (Palma and Ruiz i Altaba, 2004
). It is therefore possible that in both cases, the range of effective Shh and EGF concentrations determines the neurogenic niche where stem cells exist and where the population of early precursors can expand.
The finding that Shh
mRNA is detected in the walls of the lateral ventricles supports the idea that this molecule contributes to the definition of stem cell niches in the adult brain. However, since we have not yet been able to detect expression of Shh in sorted SVZ cell populations or in isolated single cells (not shown), it remains unclear which cells are the signaling cells. One can therefore not reject the possibility that Shh may be produced at a distance and transported through axonal terminals or dendritic arbors that reach the SVZ from afar. This possibility is also suggested, in part, by the finding that dopaminergic neurons in the ventral midbrain control SVZ cell proliferation at a distance (Höglinger et al., 2004
). Whether these cells exert their action through the secretion of Shh remains to be determined. A similar scenario has been proposed for the control of cell proliferation in the hippocampal stem cell niche, where Shh has been proposed to be transported from the septum to the subgranular layer (Lai et al., 1993). However, we note that there are cells that express Shh
in the hilus (Dahmane et al., 2001
), which could also exert a local effect. Shh has also been shown to be axonally transported in the fly visual system (Huang and Kunes, 1996
), and it is possible that it is secreted from Purkinje cell dendrites to affect external germinal layer cell proliferation in the cerebellum (Dahmane and Ruiz i Altaba, 1999
; Weschler-Reya and Scott, 1999
; Wallace, 1999
How Shh signaling is integrated with other niche factors, such as APP (Caillé et al., 2004
), is not yet clear. For instance, BMP and Shh signaling show an antagonistic relationship in the neural tube patterning (Liem et al., 1995
). In the postnatal brain SVZ, BMP signaling inhibits neurogenesis and promotes glial differentiation: ependymal cells secrete the BMP antagonist Noggin, thereby creating a favorable microenvironment for neurogenesis to occur (Lim et al., 2000
). It is possible that in the postnatal and adult SVZ, as in the embryonic neural tube, BMP and Shh signaling act antagonistically, and inhibition of the former is necessary for the latter to act.
Our results showing that Shh is a niche factor that regulates the number of SVZ cells with stem cell properties and neurogenesis, parallel those in other CNS regions: Shh
is expressed in the septum and the hilus of the hippocampus (Dahmane et al., 2001
; Lai et al., 2003
; Machold et al., 2003
), it regulates cell proliferation in the subgranular layer (Lai et al., 2003
; Machold et al., 2003
), and is also involved in the control of stem cell behavior in the developing neocortex (Palma and Ruiz i Altaba, 2004
). Shh-Gli signaling may thus be a general mechanism for the regulation of the number of stem cells and the number of precursors derived from primary progenitors. Moreover, it is interesting to propose that the control of the production of new adult neurons, from stem cell astrocytes in the SVZ and hippocampus (Doetsch et al., 1999
; Seri et al., 2001
), is largely regulated by already existing cells, located nearby or at a distance, through the action of Shh (see also Dahmane et al., 2001
), providing a mechanism for the homeostatic regulation of neuronal number and perhaps a mechanism for response to injury and disease.
Finally, our findings also suggest a method for manipulating stem cell lineages for the generation of new neurons through the regulation of Shh signaling. Such a method may help develop new strategies for the treatment of neurodegenerative diseases, such as Parkinson’s disease (reviewed by McKay et al., 2004
), by expanding stem/precursor cell population in vitro, prior to reintroduction in vivo or by the activation of dormant endogenous stem cell activity in vivo.