TLX has been shown to be essential in neural stem cell maintenance and self-renewal in adult brains9
. To uncover the molecular mechanisms involved, we have shown here that TLX activates the canonical Wnt/β-catenin signalling in adult neural stem cells. Wnt/β-catenin in turn regulates neural stem cell proliferation and self-renewal in the presence of EGF and FGF. Our observations that both Wnt7a and a constitutively active β-catenin rescued TLX
siRNA-induced proliferation deficiency in neural stem cells strongly support the notion that TLX acts in part by activating the Wnt/β-catenin pathway to regulate neural stem cell proliferation and self-renewal.
Wnt signalling has been shown to regulate the self-renewal of multiple types of stem cell. The role of Wnt signalling in the expansion of stem cells in developing brains is supported by studies in several mouse models27,29,30
. Our study demonstrated that the Wnt/β-catenin signalling is also important for adult neural stem cell proliferation and self-renewal. Wnt signalling has multiple functions in stem cells: in addition to its essential role in neural stem cell proliferation and self-renewal, Wnt signalling has also been shown to regulate neuronal differentiation of neural precursor cells42–44
. How stem cells respond to Wnts is likely to depend not only on cell-intrinsic properties but also on their specialized microenvironment45
. It is possible that Wnt/β-catenin signalling stimulates neural stem cell proliferation and self-renewal in the presence of mitogens and promotes neurogenesis on receiving differentiation cues. The presence of multiple Wnts also allows various outcomes of Wnt signalling. The Wnt pathways are therefore poised at a critical crossroads in balancing neural stem cell self-renewal versus differentiation.
The ability of TLX to repress transcription of the genes encoding intracellular molecules, such as GFAP9
and p21 (ref. 11
), indicates that TLX is a cell-autonomous regulator of neural stem cell self-renewal. Regulation of a secreted Wnt-encoding gene by TLX suggests that TLX may also function through an autocrine/paracrine mode in neural stem cells, in addition to its cell-autonomous actions. In this sense, our results support a novel concept that neural stem cells can stimulate their own proliferation by secreting signalling molecules, in addition to garnering support from other cell types, such as endothelial cells34
, in the progenitor niche. The difficulty in culturing neural stem cells at a low cell density supports the concept that neural stem cell growth requires stimulation by autocrine effect or by cell–cell contact. The fact that CCg, a molecule secreted by neural stem cells, stimulates neural stem cell proliferation and self-renewal47
lends additional support to this concept.
TLX has been shown to function as a transcription factor to repress the expression of its downstream target genes, such as p21. However, we show here that TLX activates Wnt7a expression. This activation could occur by direct binding to the Wnt7a
promoter or by repression of a Wnt7a
repressor. Binding of TLX to the consensus sites in the Wnt7a
promoter has been observed in our study, although this does not exclude the possibility that TLX also activates Wnt7a
by repressing a Wnt7a
repressor. An explanation for the activation of Wnt7a
by TLX binding could be the fact that DNA can act as an allosteric regulator to provide gene-specific regulation, the best-studied example of which is the glucocorticoid receptor48
. Our TLX
-binding-element swap experiment suggests that a specific configuration of the TLX response elements may dictate TLX to be a Wnt7a
A substantial decrease in the number of neural stem cells retaining BrdU label was detected in the subventricular zone of both Wnt7a−/− and TLX−/− adult brains, providing strong evidence that TLX–Wnt signalling regulates the neural stem cell population in the adult brain. The decrease in the number of cells retaining BrdU label in the subventricular zone of adult TLX−/− mice is more severe than that in Wnt7a−/− mice. This discrepancy could be explained by the fact that TLX, as a transcription factor, regulates a spectrum of downstream target genes. Other functional targets, in addition to Wnt7a, may contribute to the substantial depletion of neural stem cell pool in TLX−/− brains. Alternatively, the possible existence of a Wnt7a-independent stem cell population may also provide an explanation for the less severe decrease in neural stem cell numbers in Wnt7a−/− brains.
Stem cell technology holds great promise for the treatment of many diseases that currently lack effective therapies. Identifying factors that control stem cell maintenance and self-renewal is an important step in moving stem cell technology from bench to bedside. Our finding that TLX activates Wnt to stimulate neural stem cell self-renewal suggests that purified Wnt proteins or molecules activating Wnt signalling can be developed as tools to facilitate the expansion of neural stem cells as a source for cell replacement therapies in the treatment of neurodegenerative diseases and brain injury.