The olfactory bulb (OB) is one of the regions in the mammalian forebrain in which neurogenesis persists throughout life. During both development and adulthood the expression of specific transcription factors (TFs) defines different progenitor territories and specifies the formation of neuronal subtypes. Although both cell-intrinsic programs and external signals within neurogenic niches control the progression of neural stem cells (NSCs) to mature neurons, in this review we focus on the regulation of OB neurogenesis by the action of specific TFs such as Pax6, Arx, Gsx2, Tbr1, and Nurr1. We discuss how these regulatory factors and epigenetic mechanisms of gene regulation influence distinct steps of embryonic and adult neurogenesis, including their involvement in NSC proliferation and lineage differentiation, as well as in the migration of neuroblasts and the integration of newborn neurons in the synaptic network. Studies on gliogenesis have also been reviewed although there are fewer than those on neurogenesis. Much of the information presented in this review is derived from studies on the rodent OB, lateral ganglionic eminences (LGE) and subventricular zone (SVZ). When applicable, we also emphasize that the findings from rodents could be partially extended to primates, including humans, as genetic and clinical studies show that mutations in some TFs provoke alterations during human development.
NEUROGENESIS AND GLIOGENESIS IN THE EMBRYONIC OB
The development of the mouse OB starts on embryonic day (E) 10.5, although this structure cannot be detected macroscopically as an evagination of the rostral telencephalon until E12.5 (Hinds, 1968a). At E12.5, the OB can initially be divided into a ventricular zone (VZ) formed by neuroepithelial (NE) cells aligned in a radial orientation and a thinner mantle zone (MZ). These NE cells are proliferating cells that give rise to radial glia (RG) cells and to the post-mitotic neurons that form the superficial MZ (Bailey et al., 1999). Both, the NE and RG cells can be considered NSCs, also known as primary progenitors (Puche and Shipley, 2001; Vicario-Abejon et al., 2003; Kriegstein and Alvarez-Buylla, 2009; Vergaño-Vera et al., 2009).
In the OB, glutamatergic neurons are made up of mitral and tufted neurons. Mitral cells are projection neurons that arise between E10.5-E15.5, their generation peaking at E12.5, while tufted neurons include a variety of subtypes, both projection and local neurons, and they are mainly formed between E10.5 and postnatal day 0 (P0) (Fig. 1A,B,E) (Hinds, 1968a; Blanchart et al., 2006; Mendez-Gomez et al., 2011; Winpenny et al., 2011). In contrast to OB glutamatergic neurons, the production of the local GABAergic interneurons, the granule cells and periglomerular neurons (PGNs), starts from E12.5 and it is not completed during embryonic development but continues throughout the postnatal-adult life (Fig. 1E) (Hinds, 1968a; Bovetti et al., 2007; Lledo et al., 2008; Vergaño-Vera et al., 2009). The majority of the embryonic GABAergic interneurons are supplied by the dorsal LGE (dLGE) and the SVZ of the lateral ventricle between E13.5-E18.5 (Wichterle et al., 1999; Tucker et al., 2006; Vergaño-Vera et al., 2006), although the septum is also thought to give rise to OB interneurons, including calretinin (CR) cells (Long et al., 2003; Kohwi et al., 2007). Moreover, studies by Vergaño-Vera et al. (2006) revealed an endogenous population of NE cells with NSC properties (self-renewal and multipotentiality) that give rise to GABAergic and dopaminergic interneurons within the OB at E13.5 (Fig. 1A,B).
The organization of the different types of OB cells into layers is accomplished between E12.5-E19.5. After E12.5 the OB acquires a bulbar shape, and two new layers between the above-mentioned VZ and MZ can be clearly distinguished: the subependymal (SEZ) and the intermediate zone (IZ). The mitral cell layer (MCL) is first detected at E14.5, containing the mitral neurons and a few yet variable number of granule neurons. The primordia of the external plexiform layer (EPL), the glomerular layer (GL) and the olfactory nerve layer are observed between E13.5-E14.5 (Hinds, 1968b; Blanchart et al., 2008). Although by E15.5-E16.5 the IZ can no longer be detected, the primitive granule cell layer (GCL) and the internal plexiform layer (IPL) begin to develop at this time. All these processes occur with a delay of approximately 2 days in the rat (Bailey et al., 1999). From P0, all the layers that are present in the adult OB can be distinguished (Hinds, 1968b; Hurtado-Chong et al., 2009).
In the forebrain, the generation of glial cells has been reported to start at early embryonic stages (Hinds, 1968a; McCarthy et al., 2001; Kessaris et al., 2006; Parras et al., 2007; Watatani et al., 2012). However, the main wave of astrocyte and oligodendrocyte formation occurs between the end of the embryonic and the beginning of the postnatal period (Kessaris et al., 2008).
There are few studies that have addressed the development of the primary olfactory centers in humans due to the difficulty in accessing human embryonic and fetal material. In the 16th week of gestation, the OB in the human fetus has a clear ventricle and it can be divided into four zones: a periventricular cellular zone (VZ and SVZ), an intermediate zone densely populated by small cells (the IPL), a zone containing larger cells (the rudimentary MCL) and a marginal zone with a more diffuse distribution of cells (the EPL). In the 20–22 week old fetus, the ventricle persists in the caudal zone of the OB and areas of glomerulus formation appear that are more mature and more numerous in the dorsal aspect of the OB. At this stage, individual cells distributed throughout the OB are positive for S-100 protein; these cells are astrocyte precursors that have not yet acquired the characteristic shape of differentiated astrocytes. In the human fetus at 15–16 and 20–22 weeks of development, staining with synaptosomal-associated protein-25 (SNAP-25) to detect synapse formation produces a weak reaction at the periphery of the OB and in the glomeruli. Later, at 28–29 weeks, these regions show more intense SNAP-25 staining, suggesting that the primary olfactory center in humans start to function no earlier than the 30th week of development (Kharlamova et al., 2009).
NEUROGENESIS AND GLIOGENESIS IN THE POSTNATAL-ADULT OB
In the OB of rodents, interneuron generation continues in the early postnatal and adult stages (Fig. 1C–E) (Luskin, 1993; Lois and Alvarez-Buylla, 1994). The NSCs from the SVZ provide neuroblasts that migrate into the OB along the rostral migratory stream (RMS), where they differentiate into granule cells, PGNs and a subset of glutamatergic interneurons (Fig. 1C,D) (Merkle et al., 2004; Ventura and Goldman, 2007; Brill et al., 2009; Winpenny et al., 2011). The different subclasses of PGNs are generated in an age-dependent manner and while the calbindin (CB) expressing cells are preferentially produced during early postnatal life, the production of neurons that express CR and tyrosine hydroxylase (TH) is moderate in newborn animals but proceeds more rapidly during adulthood (Lemasson et al., 2005; De Marchis et al., 2007). In addition, these distinct cell types are generated in different subdomains of the SVZ (Kelsch et al., 2007; Merkle et al., 2007; Young et al., 2007; Fernandez et al., 2011; Weinandy et al., 2011). These newly added cells must be integrated into the OB circuits, and it has been reported that a number of factors regulate the synaptic integration and survival of OB neurons born in the postnatal-adult SVZ, including insulin-like growth factor-I and the microRNA miR-132 (Hurtado-Chong et al., 2009; Pathania et al., 2012).
Similar findings regarding neuroblast generation in the SVZ and their migration through the RMS to the OB have been described in primates (Kornack and Rakic, 2001; Gil-Perotin et al., 2009). The existence of immature neurons migrating to the OB has also been demonstrated in human infants, although it declines after 18 months of age and it is barely detected in adults (Sanai et al., 2004; Curtis et al., 2007; Kam et al., 2009; Sanai et al., 2011; Wang et al., 2011). However, these neuroblasts do not form a clear migratory stream but conversely, they arise and migrate as single cells at a very much lower frequency than in the prenatal period (Sanai et al., 2011; Wang et al., 2011). In fact, by measuring the levels of nuclear bomb test-derived 14C in genomic DNA it was found that there is a minimal addition of new neurons in the OB after the perinatal period in humans (Bergmann et al., 2012). However, this phenomenon could be modulated by environmental conditions which could foster neurogenesis in the adult human OB. In addition, a number of studies support the existence of local NSCs with the potential to produce granule cells and PGNs in vivo within the rodent and human OB (Fig. 1C,D) (Fukushima et al., 2002; Gritti et al., 2002; Liu and Martin, 2003; Bedard and Parent, 2004; Cave and Baker, 2009; Giachino and Taylor, 2009; Vergaño-Vera et al., 2009; Marei et al., 2012; Moreno-Estelles et al., 2012). However, this concept needs further support with additional findings about the location and possible function of the OB endogenous NSCs in adult neurogenesis.
Regarding gliogenesis, the neonatal OB is an active site of oligodendrocyte and astrocyte production, generated locally in the rostral extension of the ventricular wall (Spassky et al., 2001; Mendez-Gomez et al., 2011). In addition, NG2 expressing progenitors coming from the SVZ along the RMS also give rise to oligodendrocytes in the postnatal OB (Aguirre and Gallo, 2004).
To maintain the size of the adult OB throughout life, the continuous generation of adult-born neurons must be counterbalanced by programmed cell death in neurogenic regions of the OB (Biebl et al., 2000; Yamaguchi and Mori, 2005). Continuous neurogenesis appears to be required for the maintenance and reorganization of the whole interneuron system (Imayoshi et al., 2008). Indeed, in specific areas of the OB and SVZ there are mechanisms (growth factor and receptor homeostasis, synaptic activity, the rate of SVZ progenitors proliferation, etc.) that regulate the neuronal turnover (Hurtado-Chong et al., 2009; Murata et al., 2011; Sui et al., 2012). Cell fate and time-lapse in vivo studies in mice (Ninkovic et al., 2007; Adam and Mizrahi, 2011) show that the addition and loss of dopaminergic neurons in the GL increases over time, and that the number of newborn neurons is higher than the loss of dopaminergic neurons. This suggests the existence of a net increase in the size of this particular population with age (between 3 months and 1 year). This turnover of dopaminergic cells is dynamically controlled by the olfactory input (Sawada et al., 2011).
The functional importance of adult neurogenesis in the OB is still a matter of intense research (Lazarini and Lledo, 2011). It has been proposed that this neurogenesis is necessary for the structural preservation of the OB circuitry (Imayoshi et al., 2008) and in fact, several studies suggest that continual integration of adult-born neurons is beneficial for plasticity and odor discrimination (Lledo et al., 2006). This plasticity appears to be maximal during the initial neuronal development and it is progressively lost as the cells mature (Nissant et al., 2009). Indeed, the synapses of new neurons have a high degree of structural plasticity during a time window when they are initially integrated into the circuit but once they mature, their responsiveness to activity dependent mechanisms becomes more limited (Kelsch et al., 2009). In contrast to these findings, it was recently demonstrated that both adult-born PGNs and granule neurons undergo experience-dependent plasticity well after they have matured and become integrated into the network (Livneh and Mizrahi, 2011). Remarkably, these newborn neurons play an important role in learning processes (Alonso et al., 2012) as they are required for olfactory memory and may regulate pheromone-related behaviors, such as mating and social recognition (Feierstein et al., 2010; Sultan et al., 2010; Alonso et al., 2012).
ROLE OF TRANSCRIPTION FACTORS IN THE REGULATION OF OB NEUROGENESIS
In the developing and postnatal telencephalon the expression of TFs follows a determined patterning, and the regional combinatorial code of expression results in the generation of different types of neurons (Guillemot, 2007). We refer the readers to Tables 1 and 2 for a summary of the different TFs discussed in this article and their functional roles in OB neurogenesis. Much of the knowledge of the actions of TFs comes from the analysis of knockout (KO) mice. It should be noted that although the deletion of a specific TF could cause defects in both the OB and the olfactory epithelium (OE) (Yoshida et al., 1997; Levi et al., 2003; Long et al., 2003; Yoshihara et al., 2005; Hirata et al., 2006b; Shaker et al., 2012), we have focused our review on those TFs which are important for proliferation, specification, migration, differentiation/maturation and survival of OB progenitors and/or neurons.
Table 1. Summary of TF involved in the regulation of OB neurogenesis
Only are indicated the TFs discussed in this review.
Tbr1, Tbr2, Tbx21
Er81, Pea3, Erm
Table 2. Overview of the functions of specific TFs during OB neurogenesisa
Role in OB neurogenesis
Please, see the text for references.
Specification and survival
Mitral progenitor cells
Granule cells and PV+ interneurons
Neural stem cells
Specification and survival
MCL, EPL, GL
MCL, EPL, GL
Mitral and tufted neurons
Formation of dendrodendritic synapses
Mitral and tufted neurons
Glutamatergic, CR+ and CB+ neurons
GAD67+ and TH+ interneurons
Neuronal differentiation/ maturation
TH+ and non-dopaminergic interneurons
MCL and GL
Neuronal survival and/or differentiation
MCL and GL
The Homeobox Genes
Homeobox genes (e.g., the Paired, Gsx, Dlx and Emx gene families) control many cellular processes including proliferation, differentiation, apoptosis, cell shape, cell adhesion and migration. They are characterized by a 60 amino acid homeobox domain (or homeodomain) that is responsible for DNA-binding, although they often contain other motifs that can contribute to DNA and/or co-factor binding and that may further define their target gene specificity. These additional motifs, as well as variations in the homeodomain, are used to divide the homeoprotein superfamily into families and subfamilies (Wigle and Eisenstat, 2008).
The Paired family
The Paired family of homeobox genes are thought to be important regulators of the development of the central and peripheral nervous systems (Wigle and Eisenstat, 2008). These genes are divided into three subfamilies: the Pax- or Paired-box, the Orthodenticle and the Aristaless-related genes (Galliot and Miller, 2000).
The Paired-box subfamily
The Paired-box (Pax) TFs are involved in organogenesis and they play important roles in stem cell maintenance (Lang et al., 2007). Nine members of this TF subfamily have been identified and while all Pax TFs are expressed in the brain, with the exception of Pax1 and 9, only Pax6 mRNA is detected in the telencephalon (Stoykova and Gruss, 1994). Therefore, in this review we will focus on the activity of Pax6 in OB neurogenesis.
In the developing telencephalon, Pax6 is expressed in the pallium and it is involved in the establishment and/or maintenance of the pallial-subpalial boundary (PSB) (Carney et al., 2009; Cocas et al., 2011). In addition, Pax6 is involved in the formation of the olfactory placode, the OB and the olfactory cortex during the developing olfactory system (Stoykova and Gruss, 1994). Indeed, it was first reported that the OB does not develop in Pax6 homozygous (Sey/Sey) mutant mice, where this system is nearly absent (Dellovade et al., 1998). In contrast, the formation of an ectopic OB-like structure located at the rostral end of the telencephalon has been detected in homozygous mutant mice and rats (Jimenez et al., 2000; Hirata et al., 2002; Nomura and Osumi, 2004). In humans, heterozygous mutations in Pax6 result in aniridia and forebrain abnormalities (Sisodiya et al., 2001; Ellison-Wright et al., 2004).
In the embryonic OB, this TF is strongly expressed in both neuroepithelial cells and some postmitotic neurons (Vergaño-Vera et al., 2006; Long et al., 2007). Indeed, Pax6 is implicated in the regulation of NSC self-renewal, neural proliferation and differentiation (Sansom et al., 2009; Gomez-Lopez et al., 2011) although no studies have yet been published on the role of Pax6 in NSCs of the OB. Postnatally, the expression of Pax6 mRNA and protein is detected in the dopaminergic/GABAergic PGNs as well as in parvalbumin (PV)- and CR-positive cells located in the EPL (Stoykova and Gruss, 1994; Dellovade et al., 1998; Baltanas et al., 2009; Haba et al., 2009; Hurtado-Chong et al., 2009). A correct dose of Pax6 is essential for the migration of mitral cell progenitors (Nomura and Osumi, 2004) as well as for the differentiation and/or maintenance of specific olfactory interneurons (Nomura et al., 2007). Indeed, in the postnatal and adult OB, it is required for the differentiation of granule cells, for the specification and survival of dopaminergic PGNs, and for the differentiation and/or maintenance of PV-positive interneurons in the EPL (Hack et al., 2005; Kohwi et al., 2005; Brill et al., 2008; Haba et al., 2009; Ninkovic et al., 2010). Supporting this role, the overexpression of Pax6 in adult neuronal progenitors increases the number of TH-positive interneurons (Hack et al., 2005).
It was recently shown that the expression of Pax6 is controlled by miRNAs. Although Pax6 mRNA transcription is widespread along the ventricular walls, Pax6 protein is restricted to the dorsal aspect. This dorsal restriction appears to be due to post-transcriptional regulation in the NSCs by miR-7a, which is expressed in a Pax6-opposing ventro-dorsal gradient. Moreover, in vivo inhibition of miR-7a in Pax6-negative regions of the lateral wall induced Pax6 protein expression and increased dopaminergic neurons in the OB (de Chevigny et al., 2012a).
The Aristaless-related homeobox gene subfamily
The Aristaless-related homeobox (Arx) gene encodes one of the three largest classes of the Paired homeoproteins. During mouse development, Arx can first be detected in migrating cells within the RMS, and in the GCL and GL of the OB, at E14.5 (Yoshihara et al., 2005). It has been proposed that Arx plays an important role during embryonic central nervous system (CNS) development, and in humans, mutations in Arx appear to be responsible for brain disorders such as lissencephaly (Kitamura et al., 2002; Friocourt and Parnavelas, 2010). At P0, in the GL, TH-positive PGNs express Arx, whereas in the GCL, the majority of cells expressing Arx at E14.5 is devoid of GABA, probably because they are immature granule cells (Yoshihara et al., 2005). However, in the adult, the expression of Arx is restricted to regions that are known to be rich in GABAergic neurons, such as the OB (Poirier et al., 2004; Yoshihara et al., 2005).
Arx deficient mice die in the neonatal period, and the MCL of their OB is small and disorganized (Kitamura et al., 2002; Yoshihara et al., 2005). In these mutant mice, interneuron progenitors proliferate less and there is a defect in the entry of these progenitors into the OB, resulting in their accumulation in the RMS. In addition, the TH expressing PGNs are completely absent from the OB and the RMS of Arx deficient mice, indicating that Arx is necessary for the differentiation of these interneurons. Furthermore, these mice develop an abnormal axonal projection of olfactory sensory neurons from the OE to the OB, implicating that Arx is also required to establish functional olfactory neural circuits (Yoshihara et al., 2005).
The Genomic screened homeobox family
The Genomic screened homeobox (Gsx, formerly known as Gsh) genes are expressed in gradients throughout the embryonic ventral telencephalon. While Gsx2 protein is expressed in a dorsoventral gradient, with its highest expression in the LGE, Gsx1 is expressed in a ventrodorsal gradient, it being most strongly in the medial ganglionic eminence (Toresson et al., 2000; Vergaño-Vera et al., 2006; Wang et al., 2012).
The Gsx TFs, Gsx1 and Gsx2, have been shown to be important in patterning the ventral forebrain. In the LGE, Gsx2 expression has been implicated in the control of the PSB by repressing the expression of dorsal genes, such as Pax6. Indeed, loss of Gsx2 function provokes an expansion of this dorsal marker and a loss of interneuron migration to the OB, whereas Gsx2 overexpression in transgenic mice reduces the expression of pallial markers in the embryonic telencephalon (Toresson et al., 2000; Yun et al., 2001; Carney et al., 2009; Waclaw et al., 2009).
Furthermore, Gsx1 and Gsx2 function together in generating most of the striatum and local circuit neurons of the OB. However, while no obvious defects in striatal or OB development were detected in Gsx1 KO embryos, Gsx2 deficient embryos suffer from an early misspecification of precursors in the LGE that disrupts striatal and OB development (Corbin et al., 2000; Toresson and Campbell, 2001). In the OB, deletion of Gsx2 reduces the number of glutamic acid decarboxylase (GAD) 67 positive interneurons and augments the presence of Tbr1 positive projection neurons (Corbin et al., 2000; Toresson et al., 2000; Toresson and Campbell, 2001; Yun et al., 2003, 2001; Waclaw et al., 2009). In fact, it seems that all subtypes of OB interneurons, at least at embryonic time points, require Gsx2 for their normal production and a maintenance (Wang et al., 2009). Moreover, Gsx1/2 double homozygous mutant mice display more severe disruptions than were observed in the Gsx2 mutant alone, with an absence of OB interneurons in these double mutants suggesting that Gsx1 and Gsx2 cooperate to regulate interneuron development (Yun et al., 2003).
In addition, it has been proposed that the levels of Gsx2 regulates the quiescent and the undifferentiated state of NSCs and progenitors by inhibiting the self-renewal of NSCs, as wells as by maintaining NSC-derived progenitors in the cell cycle (Pei et al., 2011; Mendez-Gomez and Vicario-Abejon, 2012). Therefore, sustained overexpression of Gsx2 prevents cultured NSCs and progenitors to differentiate into neurons and glia in a cell-dependent manner where the NSCs isolated from the ganglionic eminences are more affected than those obtained from the OB. In vivo, Gsx2 expression produces a decrease in the number of Pax6+ and doublecortin+ neuroblasts indicating that this TF negatively regulates neurogenesis from early postnatal progenitors (Fig. 2) (Mendez-Gomez and Vicario-Abejon, 2012). The role of Gsx2 in oligodendrogenesis appears to be more complex. In cultured NSCs isolated from the OB (OBSCs), Gsx2 inhibited the formation of O4-positive oligodendrocytes. In contrast, the overexpression of this TF in the early postnatal OB produced an increase in the number of Olig2-positive cells (Mendez-Gomez and Vicario-Abejon, 2012). Concurring with this, reduced levels of Olig2 have been detected in the LGE of Gsx2 KO mice (Wang et al., 2012). All these findings suggest that the role of Gsx2 during oligodendrocyte formation may be modulated by the cell context and/or that this TF maintains Olig2-positive cells in a progenitor state which would progress to a differentiated/mature oligodendrocyte upon the action of additional factors.
The Distalless gene family
There are six murine Distalless (Dlx) genes that are organized in three bigene clusters (Dlx1/2, Dlx3/4 and Dlx5/6), although only Dlx1, Dlx2, Dlx5 and Dlx6 are expressed in the developing CNS, particularly in the differentiating GABAergic projection neurons and interneurons (Panganiban and Rubenstein, 2002). Interestingly, these two clusters (Dlx1/2 and Dlx5/6) have been associated with human autism spectrum disorders (Hamilton et al., 2005).
In general, Dlx1 and Dlx2 expression precedes that of Dlx5 and Dlx6 (Eisenstat et al., 1999). In the embryonic OB, Dlx1 and Dlx2 TFs are nearly absent although Dlx2 is expressed by cells in primary cultures of the E13.5 OB (Vergaño-Vera et al., 2006; Campbell et al., 2011). In the adult forebrain, Dlx1 and Dlx2 mRNA are expressed abundantly in the lateral ventricle, the RMS, and in the OB, in particular in PGNs and granule cells (Bulfone et al., 1998; Brill et al., 2008). By contrast, neither Dlx5 nor Dlx6 mRNAs are detected within the adult SVZ, although their expression commence at relatively low levels within the RMS and become more abundant in the OB (Brill et al., 2008). During development, Dlx1/2 positive precursors coming from both the dLGE and the SVZ of the lateral ventricle that are destined to the GL give rise to TH or CB-expressing interneurons, while those destined to the EPL give rise to PV-expressing cells. Postnatally, these Dlx1/2 positive precursors predominately differentiate into CR-expressing GL interneurons (Batista-Brito et al., 2008).
No abnormalities have been detected in the forebrain of Dlx1 mutant mice (Anderson et al., 1997), whereas disruption of Dlx2 results in a failure of dopaminergic subtype specification during OB neurogenesis (Qiu et al., 1995). In fact, Dlx2 overexpression promotes the acquisition of a dopaminergic neuronal fate during both development and adulthood (Brill et al., 2008). Although Dlx1/Dlx2 mutant mice die within a few hours of birth, it has been possible to observe that the combined disruption of these TFs results in the loss of GABAergic and dopaminergic neurons due to defects in neuronal migration and maturation. Hence, the expression of Dlx1 and Dlx2 appears to be required during OB interneuron differentiation (Anderson et al., 1997; Bulfone et al., 1998; Long et al., 2007, 2009).
In the postnatal SVZ, the expression of Dlx2 mRNA appears to be restricted to its lateral periventricular domain (Brill et al., 2008). However, Dlx2 expression is switched on in RMS neuroblasts arising from the dorsal SVZ and it is maintained in the OB, where these neuroblasts differentiate into dopaminergic neurons (de Chevigny et al., 2012b). Probably, these changes are due to the action of miR-124, which regulates Dlx2 translation during cell differentiation. In fact, miR-124 is up-regulated during the transition from transient amplifying cells to proliferating neuroblasts (which express Dlx2) in the adult SVZ, and it is further up-regulated in immature neurons from the RMS and OB (Cheng et al., 2009). Pax6 also appears to be required for Dlx2 function because the deletion of Pax6 blocks Dlx2-mediated PGN specification (Brill et al., 2008).
The Dlx5 and Dlx6 TFs are expressed at early stages in the primordial brain precursor areas of the OB and olfactory tubercle, and in the GE (Simeone et al., 1994; Campbell et al., 2011). At E16.5, Dlx5 is expressed in cells of the OB located throughout the ventricular layer and GL, and at P0 it is also expressed in juxtaglomerular neurons. In newborn animals, Dlx5 is also present throughout the RMS (Levi et al., 2003). In the adult OB, Dlx5 and Dlx6 are detected in the GCL, in the deeper granule cells of the MCL and in the GL (Allen et al., 2007). Indeed, progenitors of the Dlx5/6 lineage contribute to the CR, CB and dopaminergic subtypes of interneurons in the GL, as well as to PV-positive interneurons of the EPL (Allen et al., 2007; Kohwi et al., 2007; Li et al., 2011).
Inactivation of the Dlx5 and Dlx6 genes in mice provokes severe craniofacial, axial and appendicular skeletal abnormalities, leading to perinatal lethality (Robledo et al., 2002). Moreover, the disruption of Dlx5 produces a global defect in the differentiation of OB interneurons. These mice have a smaller OB and a disorganized MCL, produced in a non-cell-autonomous manner, and they also lack juxtaglomerular interneurons (TH- and GAD67-positive cells) and CR-positive cells in the OE (Levi et al., 2003; Long et al., 2003). In addition, Dlx5 deficient mice lack axonal connections between the OE and OB, as seen in Emx2 mutants (see below). Therefore, Dlx5 appears to be required to establish OB cytoarchitecture, for interneuron differentiation, and for the connectivity of olfactory receptor neuron axons with their targets in the OB (Levi et al., 2003; Long et al., 2003). Recent work indicates that the action of Dlx5 and Dlx2 could be in part mediated through direct activation of Wnt5a that promotes GABAergic differentiation (Paina et al., 2011).
The Emx gene family
The Emx genes are related to the Drosophila gene Empty spiracles and both Emx1 and Emx2 members of Emx gene family are expressed in the developing forebrain (Cecchi et al., 2000). While Emx1 and Emx2 are also expressed in the SEZ of the OB at E15, only Emx1 is present in the MCL. Both TFs are detected at E18 in the GL and in the EPL after birth (Mallamaci et al., 1998). However, only Emx1 is expressed in the adult OB, whereas Emx2 is absent or expressed at only very low levels (Nedelec et al., 2004). Fate-mapping experiments have shown that specific subtypes of GABAergic olfactory neurons arise from Emx1+ progenitors, including the CR-expressing PGNs (Kohwi et al., 2007).
The Emx2 KO mice die postnatally and have small OBs with a disorganized MCL. In these animals the OE also fails to project to the OB. However, Emx1 mutant mice have only minor structural defects in the forebrain (Yoshida et al., 1997; Dwyer et al., 2011), yet OB development is defective in Emx1/Emx2 double mutants. The MCL, EPL and GL in the OB of such double mutants are thin and disorganized. These more severe phenotypes in Emx double mutants than in either Emx single mutant suggest that Emx1 and Emx2 cooperate to regulate multiple features of forebrain development and that the loss of one may be partially compensate for by the other, but in a nonequivalent manner (Bishop et al., 2003).
The T-box Genes
In mammals, the T-box group includes 17 genes classified into five families (Naiche et al., 2005). T-box TFs contain at least two structural and functional domains: a sequence-specific DNA-binding domain (the T-box) and a transcriptional activator or repressor domain (Bollag et al., 1994; Kispert et al., 1995). In this review, we will focus on the Tbr1 family that has been implicated in the development of the dorsal telencephalon. This family is composed of three T-box brain (Tbr) genes: Tbr1, Tbr2 (Eomes), and Tbx21 (T-bet): (Papaioannou, 2001; Hodge et al., 2012a).
The Tbr1 gene encodes a TF that is highly expressed in the forebrain and that is involved in the development of the cerebral cortex, hippocampus and OB (Bulfone et al., 1995; Puelles et al., 2000; Mendez-Gomez et al., 2011; Hodge et al., 2012a). In the developing OB, Tbr1 is expressed in postmitotic projections neurons (Bulfone et al., 1995), suggesting that this TF could be implicated in the maintenance of glutamatergic neurons in the OB (Bulfone et al., 1998). However, recent studies have also demonstrated expression of Tbr1 protein at the beginning of mitral neuron formation from proliferative cells in the OB, i.e., during the transition from dividing progenitors to postmitotic neurons (Mendez-Gomez et al., 2011). In the postnatal-adult OB, Tbr1 is downregulated during terminal differentiation of adult-born glutamatergic neurons, although it is expressed in the external tufted cells in the GL (Brill et al., 2009; Hurtado-Chong et al., 2009; Winpenny et al., 2011).
The deletion of Tbr1 in the embryonic OB produces a reduction of mitral and tufted neurons, as well as an absence of the lateral olfactory tract that is formed by the axons of the mitral neurons (Bulfone et al., 1998). In fact, it has been demonstrated that continued expression of Tbr1 in OBSC cultures inhibits astrocyte generation and promotes neuronal and oligodendrocyte formation (Fig. 3). In the OB in vivo, sustained levels of this TF also inhibited astrocyte formation from dividing progenitors while the numbers of doublecortin-positive neuroblasts and Olig2-positive cells were not significantly affected. These results suggest that a main role of Tbr1 is to repress astrocyte formation from both NSCs and dividing progenitors. As a consequence of this partial blockage in astrocyte generation, the production of neurons and oligodendrocytes from NSCs could be facilitated by Tbr1. Alternatively, Tbr1 could be necessary for both, the inhibition of astrocyte generation and the activation of neuron formation, at least in cultured NSCs (Mendez-Gomez et al., 2011).
In humans, alterations and mutations in the CASK-TBR1-RELN signaling pathway have been involved in the development of an X-linked microcephaly syndrome and autism spectrum disorders (Bailey and Aldinger, 2009; Neale et al., 2012; O'Roak et al., 2012; Traylor et al., 2012).
The Tbr2 (formerly known Eomes) gene is closely related to Tbr1 and it is also required during neurogenesis (Bulfone et al., 1999; Kimura et al., 1999; Englund et al., 2005; Mizuguchi et al., 2012). It is also involved in the development of malformative microcephaly syndromes in humans (Baala et al., 2007).
The Tbr2 TF is implicated in glutamatergic cell fate specification in the cerebral cortex and dentate gyrus (Englund et al., 2005; Hodge et al., 2012b) and the loss of Tbr2 leads to a reduction in the number of progenitor cells in the cortical SVZ (Arnold et al., 2008; Sessa et al., 2008). Tbr2 is strongly expressed in many mitral cells in the E14 OB, and in the adult, it is found in a subset of juxtaglomerular neurons, as well as in the external, middle and internal tufted cells of the EPL that express vesicular glutamate transporter 1 (VGluT1) (Mizuguchi et al., 2012). Indeed, deletion of Tbr2 produces a reduction of its size concurring with fewer OB projection neurons, mitral and tufted cells. In fact, the MCL is undetectable, provoking a disorganization of the whole OB structure (Arnold et al., 2008; Sessa et al., 2008) reminiscent of the Tbr1 KO phenotype (Bulfone et al., 1998). It was recently shown that the specific inactivation of Tbr2 in mitral and tufted cells at late embryonic stages produces changes in gene expression, such as an increase of Tbr1, as well as, a shift of VGluT subtypes from VGluT1 to VGluT2. Therefore, these results suggest that the loss of Tbr2 led to compensatory induction of Tbr1 in a cell-autonomous manner. Furthermore, this specific inactivation of Tbr2 causes a severe impairment of the formation of dendrodendritic synapses between the mitral and tufted cells and inhibitory interneurons, leading to a hyperactivation of these glutamatergic neurons by odor stimulation. Thus, Tbr2 is also required to establish functional neural circuits in the OB and to maintain the excitatory-inhibitory balance that is crucial for odor information processing (Mizuguchi et al., 2012).
In the brain, T-box 21 (Tbx21) gene is only expressed in mitral and tufted cells in the developing OB (Faedo et al., 2002; Mitsui et al., 2011), whereas in the adult it is mainly expressed in the accessory OB (Mitsui et al., 2011). A conserved sequence has been identified upstream of the transcriptional start site of the Tbx21 gene, which contains two T-box-binding sites, suggesting that Tbx21 may be regulated by T-box TFs expressed in the mitral and tufted cells, such as Tbr1, Tbr2 or Tbx21 itself (Mitsui et al., 2011). In fact, in the OB of Tbr1 deficient mice there is no Tbx21 expression and in the Tbr2 conditional KO mice there is less Tbx21 protein (Faedo et al., 2002; Mizuguchi et al., 2012).
The Proneural Basic Helix-Loop-Helix Genes
The basic helix-loop-helix (bHLH) TFs function as heterodimers with ubiquitously expressed E proteins and they bind sequences in their target genes with the general consensus CANNTG (E-boxes). These TFs have been grouped into different families on the basis of similar sequences in the bHLH domain. A particular group of these genes encodes the proneural bHLH TFs which belong to different families (Neurogenin, NeuroD and Achaete-Scute) which regulate neurogenesis in different species from invertebrates to mammals (Bertrand et al., 2002). Indeed, these TFs are involved in both initial neuronal differentiation from progenitors and the ensuing neuronal subtype specification (Gohlke et al., 2008).
The Neurogenin family
The Neurogenins (Ngns) regulate several important stages of neural development, including the selection of neuronal progenitors, specification of neuronal phenotype at the expense of glial cell fates, and the choice of neuronal differentiation programs (Bertrand et al., 2002).
The Ngn1 and Ngn2 mRNAs are expressed in overlapping patterns throughout the developing mouse nervous system (Ma et al., 1998; Fode et al., 2000). In the embryonic OB, Ngn1 and Ngn2 are coexpressed in OB progenitors specifying the identity of the glutamatergic lineage (Shaker et al., 2012). However, in the OB, Ngn1 is also expressed in the ependymal layer and SEZ at E13.5, while it is expressed in the GCL, GL and MCL in P0 and adult mice (Zhang et al., 2007; Kim et al., 2011b). The Ngn2 mRNA and protein are expressed in the OB between E11.5 and E13.5 (Osorio et al., 2010; Winpenny et al., 2011) but in the adult brain, its expression is largely restricted to the dorsal region of adult SVZ of the lateral ventricle (Brill et al., 2009).
The Ngn1 TF is involved in the migration of olfactory interneurons in part by controlling the production of prokineticin 2, a chemoattractant secreted by OB neurons (Zhang et al., 2007). Indeed, in the OB of Ngn1 KO mice, many TH-positive cells did not reach their final destination in the GL but were stalled in the GCL, demonstrating a defect in the migration of these interneurons (Zhang et al., 2007). Furthermore, only a vestigial of OB is formed in Ngn1 and Ngn2 double mutant mice and fewer glutamatergic mitral and juxtaglomerular cells are differentiated in these double mutants, supporting that both TFs cooperate during OB neurogenesis (Shaker et al., 2012).
A central role of Ngn2 has been demonstrated during OB neural subtype specification as it is involved in the lineage determination of glutamatergic neurons from embryonic progenitors and adult SVZ cells (Brill et al., 2009; Winpenny et al., 2011; Shaker et al., 2012). In addition, it has been reported that Ngn2 directs the differentiation of Achaete-scute homolog 1 (Ascl1) -expressing precursors isolated from adult SVZ progenitors into CR- and CB- expressing neurons (Roybon et al., 2009). Together, these studies show that Ngn2 has a role during interneuron differentiation in the adult SVZ-OB in addition to its major function in glutamatergic cell specification and differentiation during the embryonic period.
The NeuroD family
The NeuroD1 TF has neuronal differentiation activity and it is the downstream mediator of Ngn activity (Lee et al., 1995). NeuroD1 is expressed in OB nascent postmitotic neurons between E11.5 and E13.5 (Osorio et al., 2010), while in the adult it is detected in the GL and specifically in mature PGNs (Boutin et al., 2009). It has been shown that premature expression of NeuroD1 in vitro and in vivo induces the differentiation of forebrain progenitors (Boutin et al., 2009). In addition, NeuroD1 is sufficient to cause adult SVZ progenitors to differentiate into CR-expressing neurons (Boutin et al., 2009; Roybon et al., 2009). Conversely, knockdown of NeuroD1 induces a dose-dependent inhibition of terminal maturation of PGNs in the GL (Boutin et al., 2009; Gao et al., 2009).
The Achaete-Scute family
The proneural gene Ascl1 (formerly known Mash1) is essential for neurogenesis in the embryonic ventral telencephalon (Casarosa et al., 1999; Horton et al., 1999). In the adult, it has been demonstrated that this proneural TF is transiently expressed in all progenitors of the olfactory interneuron lineage (Parras et al., 2004; Kim et al., 2011a).
It was proposed that Ascl1 might be required for normal development of OB interneurons (Casarosa et al., 1999; Parras et al., 2004) and indeed, recent work indicates that Ascl1 controls molecular pathways acting in the specification of these interneurons (Wang et al., 2009). In Ascl1 mutants there are OB interneuron defects (Long et al., 2007), specifically, fewer OB interneurons being generated in these mice and granule cells are more severely affected than the PGNs (Casarosa et al., 1999; Parras et al., 2004; Wang et al., 2009).
The Sp/XKLF Genes
The Sp/XKLF genes are subdivided into two major families, the Sp and the KLF, both of which share the common characteristic of three conserved zinc fingers that form the DNA-binding domain (Bouwman and Philipsen, 2002). In this review, we will focus on the Sp family, and particularly on Sp8 that is expressed in neurogenic regions of the embryonic (dLGE and OB) and postnatal (SVZ, RMS and OB) brain (Waclaw et al., 2006).
The expression of Sp8 is clearly detected in the E18.5 OB and it is maintained in different populations of OB interneurons in both the GCL and GL of the postnatal-adult brain. Sp8 is expressed in almost all CR-positive cells and in many GAD65:-positive neurons, whereas it is largely absent in the CB population (Waclaw et al., 2006; Kosaka and Kosaka, 2012). Although Waclaw et al. (2006) concluded that most TH cells were Sp8 negative, two recent works report that many TH-expressing cells are Sp8 positive (Chen et al., 2012; Kosaka and Kosaka, 2012). In addition, it has been demonstrated that all PV-positive interneurons in the adult EPL also express Sp8 (Li et al., 2011; Chen et al., 2012).
Conditional inactivation of Sp8 in the ventral embryonic telencephalon produces a reduction in size of the OB (Waclaw et al., 2006). Moreover, the Sp8-expressing interneurons coming from the dLGE and SVZ are absent in the OB of Sp8 conditional mutants (Waclaw et al., 2006; Li et al., 2011). This indicates that the Sp8 is necessary for the differentiation and/or survival of the majority of OB interneuron populations.
The E-Twenty Six Genes
All E-twenty six (Ets) gene members present a highly conserved DNA binding domain, the ETS domain, which is a helix-turn-helix structure. Nevertheless, there is sufficient variability in the ETS domain of different members of the group to permit their classification into families on the basis of sequence identity. The PEA3 family is composed of three members, Pea3, Erm and Er81, which are expressed in numerous developing organs suggesting they fulfill a key role in murine organogenesis (de Launoit et al., 1997). The Pea3 TF is expressed in the SVZ and RMS but not in the OB (Cave et al., 2010). By contrast, Er81 and Erm are expressed in the OB, in cells of both the GL and MCL, although they do not co-localize. Within the GL, Er81 is present in TH positive cells and in CR interneurons (Allen et al., 2007; Saino-Saito et al., 2007). In addition, Er81 is expressed in the GCL of the OB, as well as in the dLGE, SVZ and RMS (Stenman et al., 2003).
In primary cell cultures of the OB, ectopic expression of Er81 increases the number of cells positive for the dopaminergic marker TH. In fact, there is a reduction in the number of TH expressing cells in the OB of mice lacking Er81, whereas other PGN subtypes remain unaffected or less severely reduced. However, in these mutants the dopaminergic progenitor cells in the dLGE that already express Er81 also appear to be unaltered. Therefore, Er81 could control a late stage of dopaminergic neuron differentiation in the OB (Flames and Hobert, 2009).
It has been proposed that Er81 induces only TH expression and not that the other cassette genes (Aadc, Gtpch, Dat, and Vmat2) necessary for the maturation of dopaminergic neurons in the OB. These studies also revealed that Er81 binds directly to a consensus binding site/dopamine-motif in only the rodent TH proximal promoter, suggesting that the binding of Er81 to the TH promoter follows a species-specific mechanism (Cave et al., 2010).
The Immediate Early Genes
The genes encoding orphan nuclear receptor family 4 (NR4A) TFs are considered to be Immediate early genes. Nuclear receptors have a common structure that consists of a weakly conserved amino-terminal A/B region containing the activation function-1 transactivation domain, a highly conserved DNA-binding domain, and a highly conserved carboxy-terminal ligand-binding domain. The activity of the NR4A family is regulated at the level of gene expression and protein stability, independently of its ligands. This family consists of three members, NGFI-B, Nurr1 and NOR1, which are predominantly expressed in the CNS (Hawk and Abel, 2011). In this review we will focus on Nurr1 that is essential to mediate the synaptic activity-dependent expression of TH in the OB dopaminergic precursor cells (Cave and Baker, 2009).
The Nurr1 TF is expressed in the OB in a synaptic activity-dependent manner (Saino-Saito et al., 2004), and high levels of Nurr1 expression are found in TH positive PGNs, as well as in the MCL and GCL in the adult mouse (Backman et al., 1999). The TH gene has a Nurr1-responsive element that can activate TH transcription (Sakurada et al., 1999) and in odor-deprived adult mice subjected to unilateral naris closure, Nurr1 and TH mRNA expression is downregulated concomitantly in the OB. Hence, the expression of Nurr1 would appear to be necessary for OB dopaminergic differentiation (Liu and Baker, 1999; Saino-Saito, 2008). However, TH expression is maintained in the OB of Nurr1 deficient mice (Saucedo-Cardenas et al., 1998; Le et al., 1999) suggesting that NGFI-B, which is also expressed in the OB, might compensate for the lack of Nurr1 activity in the OB of these mutant mice (Liu and Baker, 1999).
The Sal-Like Genes
The Sal-like (Sall; originally termed as Spalt in Drosophila) genes encode a family of zinc finger TFs, four of which are expressed during mammalian development. The Sall1 and Sall3 mRNAs are expressed in both the developing OB and OE (Ott et al., 1996, 2001). At E13.5, OB progenitor and differentiating cells express these TFs (Harrison et al., 2007; Harrison et al., 2008), while at E17.5 both TFs are expressed in the MCL, GCL and GL, and their expression is retained in these layers in the adult OB. However, the level of Sall1 is higher in the MCL, while Sall3 expression is stronger in the GL where this TF is detected in TH cells (Harrison et al., 2007, 2008; Heng et al., 2011). The disruption of Sall1 produces a reduction in OB size and a disorganization of the OB laminar structures. In particular, this TF is required for the laminar organization of the MCL and it regulates mitral cell neurogenesis in the developing OB in a spatial-dependent manner (Nishinakamura et al., 2001; Harrison et al., 2007).
The deletion of Sall3 produces a loss of the TH-positive interneurons and a decrease in the non-dopaminergic interneurons in the GL, suggesting that Sall3 is necessary for the terminal differentiation of TH-expressing cells, as well as for the maturation of dopaminergic and non-dopaminergic interneurons. Indeed, the precursors of these cells are present in the OB of Sall3 mutant mice (Harrison et al., 2008; Heng et al., 2011).
The Forkhead/Winged-Helix Genes
The Forkhead/winged-helix (Fox) genes have a highly conserved domain, the “winged helix,” which is composed of a helix-turn-helix core of three α-helices flanked by two loops, or “wings.” The Foxj1 TF is a member of this family that plays important roles in cilia formation in the respiratory, reproductive, and central nervous systems (Carlsson and Mahlapuu, 2002). In fact, it has been demonstrated that Foxj1 is required for multiple developmental processes in ependymal cell differentiation (Jacquet et al., 2009).
The Foxj1 mRNA is expressed in a small subset of astrocytes and immature glia in the adult SVZ and RMS (Jacquet et al., 2009). In addition, it was recently shown that the expression of Foxj1 in adult mice is restricted to the SVZ, RMS and OB, where this TF is detected in the core of the OB and in the MCL (Jacquet et al., 2011).
When Foxj1 is deleted there is a reduction in OB size, as well as a defect in all the layers, especially in the GL, which is no longer detected. In addition, the proliferation and neurogenic activity is severely disrupted in the absence of Foxj1 in vitro, as well as during OB development in vivo. Furthermore, it has been demonstrated the existence of a unique progenitor domain defined by the expression of Foxj1, which begins in the embryonic LGE and that is maintained in the rostral extension of the RMS and in the neonatal and adult OB. This domain gives rise to a confined, not yet characterized, population of OB neurons that switches off their Foxj1 expression after differentiation. However, deletion of Foxj1 affects to both Foxj1-derived and Foxj1-independent neurons, indicating the Foxj1 expression is required for the progression and regulation of embryonic to postnatal neurogenesis in the OB, both in a cell autonomous and non-autonomous manner (Jacquet et al., 2011).
The Forebrain Embryonic Zinc Finger Genes
The Forebrain embryonic family of zinc finger (Fez) TFs contains six C2H2-type zinc fingers in their carboxy terminal region and an Eh1 repressor motif in their amino terminal region. During development, two members of this family are expressed in overlapping domains in the forebrain, Fezf1 (also called as Fez or Zfp312-like) and Fezf2 (formerly known as Fezl or Zfp312). Fezf1 is normally expressed in the olfactory sensory neurons but not in the OB projection neurons, interneurons nor interneuron progenitors. Fezf2 is expressed specifically in the vomeronasal organ (Shimizu and Hibi, 2009).
In Fezf1 deficient mice, the OB is smaller and shows abnormal layer formation. As such, the MCL is thicker, the polarity of the mitral cells is disorganized and the PGNs in the GL are absent. In these mice, the migration of the interneuron progenitors through the RMS is altered and the olfactory axons stop growing before reaching the OB without completing their projection (Hirata et al., 2006b; Watanabe et al., 2009). Therefore, the role of Fezf1 in MCL formation and interneuron development in the OB is not a cell autonomous process, since this TF is not expressed in these neuronal progenitors (Hirata et al., 2006b). Moreover, disruption of Fezf1 and Fezf2 produces a loss of OB morphology that is more severe than that seen in Fezf1 mutant mice (Hirata et al., 2006a; Shimizu et al., 2010), although the precise function of Fezf2 in the OB remains unknown.
THE ROLE OF EPIGENETIC MODIFICATIONS IN OB NEUROGENESIS
Epigenetic mechanisms which include DNA methylation, histone modification and non-coding RNAs have emerged as key regulators of the gene expression that is required for NSC maintenance and fate specification. Epigenetic regulation can lead to relatively long-lasting biological effects and maintain functional neurogenesis throughout life in discrete regions of the mammalian brain (Jobe et al., 2012).
DNA methylation is a well-known mechanism underlying long-term gene silencing (Reik, 2007). During the transition of embryonic stem cells to NSCs, many genes associated with pluripotency are methylated and silenced, which highlights the importance of DNA methylation during neural induction (Mohn et al., 2008). In addition, 5-hydroxymethylcytosine (5-hmC) enrichment has been observed in NeuN-positive mature neurons in the GL of the OB, this being a DNA base implicated in DNA methylation. Indeed, co-localization of 5-hmC with mature (CB and NeuN positive) but not immature neurons suggests that 5-hmC-mediated epigenetic modification may play a role in a late step of neuronal development (Szulwach et al., 2011).
Acetylation of histones occurs at lysine residues and it is catalyzed by histone acetyltransferases (HATs). Histone acetylation is a reversible process and deacetylation is catalyzed by histone deacetylases (HDACs). As a general mechanism, histone acetylation promotes gene expression by increasing transcription-activator protein access to regulatory genomic DNA regions. By contrast, histone deacetylation compacts chromatin and represses gene expression by restricting access of transcription-activator proteins (Akiba et al., 2010). Significantly, both HATs and HDACs have recently been implicated in regulating adult neurogenesis (Sun et al., 2011). Indeed, HDAC function is critical for regulating TH expression in both neural progenitors and mature OB dopaminergic neurons. However, the different responses to the combinatorial exposure of HDAC inhibitors and depolarizing culture conditions indicate that TH expression in neural progenitors in the RMS and mature OB neurons are regulated by distinct HDAC mediated mechanisms (Akiba et al., 2010). In addition, a recent study with inducible deletion of HDAC2 in neural progenitors using GLAST-CreERT2 mice revealed an abnormal maturation of newborn neurons in the SVZ/OB system which die at a specific stage in this process. In addition, HDAC2 appears to be necessary for maintaining a normal proliferation rate in progenitor cells and for silencing the expression of Sox2 during the transition of a progenitor into a neuroblast (Jawerka et al., 2010).
The Querkopf KO mouse is another example of the consequences of acetylation. The lack of Querkopf, a MYST family transcriptional co-activator with HAT activity, promotes a reduction in the proliferation of neural progenitors in the SVZ. Similarly, its absence is associated with a decrease in the number of migrating neuroblasts in the RMS and new interneurons in the OB of middle-aged mice indicating that Querkopf is essential for normal adult neurogenesis (Merson et al., 2006).
Methylation of histone tails is another factor that determines the active or silent status of chromatin. Lysine and arginine residues in histones can be methylated by histone methyl transferases (HMT), and their methylation can produce either activation or repression of gene transcription. For example, H3K4 methylation is a marker for transcriptional activation whereas methylation on H3K9 or H3K27 is known to be associated with gene silencing (Kim and Rosenfeld, 2010). The polycomb and trithorax group (PcG and TrxG) proteins are antagonistic chromatin complexes that silence or activate their target loci, respectively, maintaining the corresponding expression state over many cell divisions. In addition, they have been implicated in regulating specific aspects of embryonic and adult neurogenesis (Ringrose and Paro, 2007; Roman-Trufero et al., 2009).
Polycomb group proteins
The PcG genes encode subunits of chromatin complexes, the polycomb repressing complexes. Two of these subunits are endowed with chromatin-modifying activities: HMT methylates a lysine of histone H3 whereas E3, which is a monoubiquitin ligase, modifies a lysine of histone H2A in a subsequent step that stabilizes transcriptional repression (Cao et al., 2002; Wang et al., 2004).
It has been proposed that the PcG protein Bmi1, which catalyzes H3K27 methylation, is a key epigenetic regulator for self-renewal and maintenance of the developmental potential of NSCs (Molofsky et al., 2003). In addition, it was recently demonstrated that the PcG histone E3 monoubiquitin ligase Ring1B is involved in the maintenance of the self-renewal capacity of OBSCs and their undifferentiated state, principally by repressing cell cycle inhibitors and through the activation of Notch signaling pathways. Furthermore, Ring1B represses Ngn1 expression preventing neuronal differentiation while favoring astrocyte generation (Hirabayashi et al., 2009; Roman-Trufero et al., 2009).
Trithorax group proteins
The TrxG proteins catalyze and maintain histone tail H3K4 methylation, resulting in stable and transcriptional active chromatin domains (Ringrose et al., 2003). One TrxG member, mixed-lineage leukemia 1 (Mll1), encodes an H3K4 HMT that is specifically required for neuronal but not glial differentiation of adult NSCs. In adult mice, Mll1 is expressed in the SVZ and OB, and it is required for proliferation and neurogenesis in these zones. Using a conditional KO “floxed” allele of Mll1 it was shown to have a strong influence on the size of the OB, probably due to the control exerted on the addition of neurons in the postnatal OB. Indeed, the loss of Mll1 provokes a decrease in the rate of neurogenesis, as well as impairing neuroblast migration. Mll1 has been proposed to bind directly to the Dlx2 promoter of NSCs in the SVZ, stimulating their neuronal differentiation (Lim et al., 2009).
Noncoding RNAs (NcRNAs) are transcribed from non-protein coding regions in the genome, and they have emerged as an important class of epigenetic regulators that interact with chromatin modifiers and TFs to regulate gene expression (Hobert, 2008). One major class of NcRNAs are the miRNAs, which inhibit gene expression through post-transcriptional mechanisms and regulate neurogenesis (Kawahara et al., 2012). In this review article, the important role of miRNAs in regulating the activity of TFs and the production of different types of neurons in the OB has been discussed in the specific subheadings dedicated to these TFs.
Neurogenesis is a complex process that requires the coordinated control of intrinsic and extrinsic regulators that act at multiple levels in a spatiotemporal-dependent manner. Substantial progress has been made in the identifying the molecular mechanisms involved in the regulation of OB neurogenesis by TFs. However, complete and sequential genetic programs controlling the development of OB neuronal lineages from NSCs, and the relative contribution of cell autonomous mechanisms and environmental signaling to cell specification and neuronal integration in the olfactory system still needs to be further characterized. Considering the relevance of adult NSCs to brain homeostasis and their potential use in cell therapy for brain repair, a deeper understanding of these processes will be crucial for the in vitro generation of specific neuronal subtypes and for the activation of endogenous neurogenesis and gliogenesis.