Introduction
The mammalian central nervous system (CNS) consists of multiple neuronal cell types, as well as glial cells, astrocytes and oligodendrocytes, which support the neuronal functions. These cells are derived from common neural precursor cells (NPCs; also known as neural stem cells, NSCs). NSCs exhibit the two essential properties of stem cells: self-renewal and multipotency. Generally, both stem cells and NSCs are able to generate these differentiated cells (
Gage, 2000;
Schuurmans and Guillemot, 2002;
Gotz and Huttner, 2005). Although both stem cells and neural stem cells are proliferating cells, there are still some differences between them. Stem cells usually have abilities to keep on self-renewing, replicating indefinitely and generating an unlimited number of progenies. Through symmetric cell division, one stem cell generates two daughter stem cells, and through asymmetric cell division, one stem cell gives rise to two different cells. Among them, one is still stem cell, owning the same potential as its mother cell and the other has a different cell fate, becoming an NSC. Similar to stem cells, NSCs are immature and proliferative cells, but their ability to generate new types of cells is limited. Through symmetric cell division, one neural progenitor cell can generate two neural progenitor cells which both are still immature and proliferating cells. In contrast, through asymmetric cell division, one neural progenitor cell gives rise to two cells. Among them, one is still NSC while the other becomes neuron and migrates towards the cortical plate (CP) along radial glial (
Noctor et al., 2004;
Sun et al., 2007).
During mammalian CNS development, NSCs are mainly accumulated in the ventricular zone (VZ) of the neural tube. NSCs are found in other areas of the neocortex in both human and mouse (
Hansen et al., 2010). Embryonic NSCs can give rise to almost all cell types required for the formation and function of the CNS. Until recently, neurogenesis was largely understood to be restricted to prenatal and early postnatal development, with no neuronal regeneration in the adult brain. In contrast to the earlier dogma, it is generally accepted now that neurogenesis occurs throughout life in mammalian brains. Neurogenesis in adult brain has been noticed at least in two locations: the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus in the hippocampus.
Neurogenesis is an extremely complicated process including proliferation, differentiation, migration and maturation in a distinct and sequential manner, accompanied by apoptosis of the nonfunctional neurons. It is regulated at different levels by both extrinsic factors, such as biophysiological and pathological conditions, and intrinsic factors, such as genetic and epigenetic programs. The maintenance/proliferation, differentiation, migration and maturation of NSCs are tightly controlled by intricate molecular networks. Uncovering these regulatory mechanisms is crucial not only for understanding the functions of the CNS but also for the application of stem cell for therapeutic benefits. Considering that the application of stem cell for clinical therapy has met the bottleneck in inducing the specific differentiation of NSC into functional neurons and the migration of those neurons to the right positions, more studies have to be concentrated on understanding the basic mechanisms in these processes in vivo. In this minireview, we will mainly focus on the role of bone morphogenetic protein (BMP) signaling in NSCs during mammalian brain development.
SVZ neurogenesis
During neocortical development, NSCs pass through sequential phases of expansion, neurogenesis, and gliogenesis (
Miller and Gauthier, 2007). During the expansion or proliferation phase, NSCs or NPCs, which are often referred to as neuroepithelial cells (NEs), expand their population by symmetric cell division that produces two NEs (
Götz and Huttner, 2005). However, around the onset of the neurogenic phase, NSC turns into radial glia (RG), which is able to self-renew and generate more differentiated cells simultaneously through asymmetric cell division. The timing of the onset of the neurogenic phase would greatly affect the neuronal number, since it regulates the initial pool size of NSCs (before starting neurogenesis).
After differentiation, neurons migrate to different layers of cortex via special routes to form a functional neural circuitry. The newborn neurons transiently become multipolar with multiple processes within the SVZ and lower intermediate zone (IZ), which is also known as the premigratory zone, and then transit to bipolar cells to migrate out of the premigratory zone along radial fibers to the CP (Fig. 1) (
Hatten, 1999;
Marín and Rubenstein, 2003;
Tabata and Nakajima, 2003;
LoTurco and Bai, 2006;
Guo and Wang, 2009). Generally, radial glial is recognized as a kind of NSC. During brain development, it is not only involved in generating new-born neurons, but also responsible for supporting and guiding the proper migration of newborn neurons (
Goldman, 2003;
Weissman et al., 2003). After the newborn neurons arrive at the proper layers of CP and further differentiate into more matured neurons, the majority of radial glial undergo transformation to become astrocytes eventually. In addition, studies from
in vitro cell culture revealed that multiple lineages of astrocyte all come from glial-restricted precursors (GRPs), which are also originated from the multipotent NSCs. GRPs can differentiate further into different cell types: type 1 astrocytes, oligodendrocytes, and type 2 astrocytes.
The BMP signaling pathway
A combination of genetic and environmental cues and a series of internal cellular signaling molecules coordinately influence the proliferation and determine the cell fate of NSCs in the developing brain. Several canonical signaling, such as Wnt, Shh, transforming growth factor (TGF)/BMP, Notch, growth factors and retinoic acid (RA) are the major pathways involved in this process (
Hirabayashi et al., 2004;
Hirabayashi and Gotoh, 2005).
BMPs including BMP2, 4, 5, 6, and 7 belong to a subclass of the transforming growth factor beta (TGF-beta) superfamily. Among these external cues and internal cellular signaling of NSCs, BMP signaling, containing BMPs and their downstream molecules, plays a critical role during NSC fate determination, in addition to the involvement in stem cell self-renewal and multipotency and tumorigenesis as discovered previously (
Chen and Panchision, 2007;
Sabo et al., 2009). In canonical BMP/SMAD signaling, BMP, acting as an extracellular ligand, transduces its signal via binding to the transmembrane receptors which are, in turn, assembled into a functional receptor complex possessing phosphorylation capability to the intracellular downstream signaling mediators, R-SMAD proteins —SMAD1, SMAD5, and SMAD8. The R-SMADs are then activated and can form a complex with the co-SMAD, SMAD4, to regulate target gene expression through cooperation with other DNA-binding factors or transcription factors in the nucleus (
Massagué and Chen, 2000;
Shi and Massagué, 2003;
Fei et al., 2010) (Fig. 2).
The SMAD-mediated BMP signaling functions mainly at the transcription level through activation or suppression of its target genes. However, BMP can also activate a series of other downstream molecules in a non-transcriptional way. For example, BMP can activate LIMK (LIM-domain containing protein kinase) or MKK3/6 (mitogen-activated protein kinase kinase 6) and activate other downstream molecules subsequently (
Shi and Massagué, 2003). The treatment of BMP4 also leads to the activation of signal transducer and activator of transcription (STAT) directly, which is in turn associated with rapamycin-associated protein (FRAP), to induce astroglial differentiation (
Rajan et al., 2003).
BMP signaling and NSC differentiation
BMPs are crucial regulators for the differentiation of embryonic stem (ES) cells as well as NSCs during development. Different groups have provided evidence that different members of BMP family can inhibit the proliferation of NSCs. In mouse neuroepithelial cell culture, BMP2 can suppress the proliferation of this kind of NSCs (
Nakashima et al., 2001). Exogenous BMP2 can also effectively inhibit the proliferation of neural progenitor cells in mouse SVZ (
Gross et al., 1996). Similarly, BMP2 and BMP4 produced by brain endothelial cells (BECs) repress the proliferation of E14.5 mouse NSCs
in vitro. In accordance with this, treatment of NSCs with Noggin, a BMP antagonist, significantly promotes the proliferation. During this process, the activation of SMAD can be detected, and the increased proliferation by silencing SMAD5 with siRNA demonstrates that the proper proliferation of NSCs is regulated by BMP/SMAD signaling (
Mathieu et al., 2008;
Sun et al., 2010).
In addition to regulating the proliferation of NSC, BMPs are also involved in the determination of NSC fate. Generally, different members of BMP family promote astroglial differentiation of NSCs both
in vivo and
in vitro (
Gross et al., 1996;
Mabie et al., 1999;
Bonaguidi et al., 2005;
Kasai et al., 2005). Mabie et al. have shown that BMP2 can promote effectively astroglial differentiation of NSCs from rat cerebral cortex (
Mabie et al., 1999). The results from other groups are also consistent with this. Gross et al. have tried to identify the role of BMPs family in cellular differentiation in the CNS and peripheral nervous system (PNS). BMP2, 4, 5, 6, and 7 were added into culture medium individually or in combination. Surprisingly, all of the BMPs effectively promoted astroglial lineage commitment of precursor cells isolated from E17 mouse SVZ. This result is consistent with the assumption that the members of BMPs family are conserved in both structure and function. It also unveiled the importance of BMPs in NSC differentiation (
Gross et al., 1996). In the cultured cells from E18.5 mouse ganglionic eminence, BMP4 or its antagonist, noggin, induces or inhibits astroglial differentiation, respectively (
Bonaguidi et al., 2005). In E14.5 mouse striatum NSCs, expression of beta-catenin can induce the expression of BMPs and astroglial differentiation. The increased astroglial differentiation can be inhibited by noggin (
Kasai et al., 2005). The above results were confirmed
in vivo later. The BMP signaling mutant mice exhibit glial cell maturation defects (
See et al., 2007) and the BMP4 transgenic showed increased astroglial lineage commitment (
Gomes et al., 2003).
Besides differentiation into astroglial, NSCs also have the potential to differentiate into oligodendrocyte cells. In contrast to induction of astroglial differentiation of NSCs, BMPs can effectively inhibit oligodendroglial differentiation both
in vivo and
in vitro (
Mabie et al., 1999;
Kasai et al., 2005;
Bilican et al., 2008). During development, endogenous cues including platelet-derived growth factor receptor alpha (PDGFR alpha), Sonic hedgehog homolog (Shh), and other intrinsic factors are activated and have been implicated in oligodendrocyte fate specification (
Bradl and Lassmann, 2010). Olig1 and Olig2, two important transcription factors involved in cell fate determination of NSCs, interact with some other regulators and further promote oligodendrocyte differentiation (
Zhou and Anderson, 2002). The treatment of NSCs with noggin or the transfection of NSCs with beta-catenin both result in an increase of oligodendroglial differentiation (
Kasai et al., 2005).
BMPs have also been proved to be involved in neuronal differentiation of NSCs (
Sabo et al., 2009). The influence of BMP2 on NSCs is complicated and depends on the cell density of NSCs and the concentration of BMP2. When cell density is low or moderate, BMP2 appears to promote neuronal differentiation. In contrast, BMP2 inhibits neuronal differentiation of subventricular zone NSCs where the cell density is high. Low concentration of BMP2 promotes neuronal differentiation, but relatively higher concentration of BMP2 inhibits neuronal differentiation. Therefore, BMPs appear to modulate neuronal differentiation depending on a series of cues (
Gross et al., 1996;
Sabo et al., 2009).
Despite considerable progress in understanding the role of BMPs in cell fate determination, the underlying mechanisms are still poorly understood. Among the target genes of BMP/SMAD signaling, inhibitors of DNA binding (IDs) were identified earlier. IDs are a subfamily of helix-loop-helix (HLH) proteins which can form heterodimers with members of the basic HLH transcription factors and inhibit the DNA binding and transcriptional activation. Several members of IDs are induced significantly in cultured NSCs by BMPs. They can form a complex with transcription factor 3 (TCF3), contributing to the generation of astrocytes. Meanwhile, the two transcription factors, Olig1 and Olig2 that can induce oligodendrocyte differentiation, are both obstructed to bind to DNA by IDs (
Samanta and Kessler, 2004). Recently, there is also evidence showing that Olig2 is the target gene of SMAD4 and is regulated by BMP/SMAD signaling directly (
Bilican et al., 2008). During cell fate determination of NSCs, RE1 silencer of transcription (REST)/neuron-restrictive silencer factor (NRSF) is identified as BMP/SMAD target gene by chromatin immunoprecipitation (ChIP). During the astrocytic differentiation of NSCs, REST/NRSF is up-regulated and sustained by BMP/SMAD signaling activation to suppress neuronal differentiation of NSCs. In addition, endogenous REST/NRSF was found to be associated with the neuronal genes and disruption of its function resulted in the derepression of those genes in differentiated astrocytes. This may explain why neuronal genes expression are suppressed when NSCs are set for differentiating into astrocytes (
Kohyama et al., 2010).
BMP signaling and brain development
Recently, injection of BMP7 into the lateral ventricule of mouse at midgestation has been shown to induce the early differentiation of radial glia into glial precursors and astrocytes. The precocious radial glia maturation leads to the defect in the laminar distribution of late-born neurons, most likely by disrupting radial glia’s ability to support neuronal migration. We have found that the BMP signaling downstream transcription factor, SMAD1 and collapsin response mediator protein-2 (CRMP2) are inversely and complementarily expressed during brain development (
Sun et al., 2010). BMPs and overexpression of SMAD1 or 4 can suppress CRMP2 expression both
in vivo and
in vitro. Knockdown of CRMP2 expression or overexpression of dominant negative forms of CRMP2 cause the accumulation of multipolar cells in the ventricular zone, subventricular zone and intermediate zone and suppresses neurite outgrowth, suggesting that CRMP2 is required for multipolar to bipolar transition for directional neuronal migration as well as neurite outgrowth. It is very likely that BMP signaling may provide a checkpoint during brain development by suppressing the expression of CRMP2 to prevent the premature development of cortical neurons (Fig. 2).
Conclusion
Although the potential of applying stem cell therapy for regenerative medicine is very promising, there is still a long way to go in our understanding of the complicated molecular networks that regulate the differentiation, migration and maturation of NSCs before the practical application of stem cell for clinical therapy.
Almost all research involving BMP signaling in CNS development is currently focused on SMAD-mediated pathway. The non-SMAD-mediated pathway has been proved to be important for embryogenesis and tumorigenesis. The importance of the non-SMAD-mediated pathway in neurogenesis is to be elucidated.
BMP family is a subclass of the TGF-beta superfamily. TGF-beta has been reported recently to regulate the specification of a single axon and multiple dendrites during brain development by modifying the phosphorylation of Par6. It seems that the TGF superfamily may have diverse functions, which remain to be explored.
In conclusion, even though studies from our group and others indicate that the BMP signaling is essential for CNS development, the underlying mechanisms remain largely unclear. Therefore, research on the molecular mechanisms of the BMP signaling in CNS development, especially in vivo studies of the role of the BMP signaling in NSC differentiation should be reinforced in the future.
Higher Education Press and Springer-Verlag Berlin Heidelberg
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