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Keywords:

  • fgf;
  • neurogenesis;
  • neuronal migration;
  • olfactory system;
  • Pax6

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Conclusions
  5. Acknowledgments
  6. References

The olfactory system is responsible for capturing and processing odorant information, which significantly influences a variety of behaviors in animals. The vertebrate olfactory system consists of several neuronal components including the olfactory epithelium, olfactory bulb and olfactory cortex, which originate from distinct embryonic tissues. The transcription factor Pax6 is strongly expressed in the embryonic and postnatal olfactory systems, and regulates neuronal specification, migration and differentiation. Here we review classical and recent studies focusing on the role of Pax6 in the developing olfactory system, and highlight the cellular and molecular mechanisms underlying the highly coordinated developmental processes of the vertebrate olfactory system.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Conclusions
  5. Acknowledgments
  6. References

In animals, the sense of smell (i.e. olfaction) influences a variety of behaviors such as feeding, self-defense, social interaction and reproduction (Hatt 2004; Bargmann 2006). Olfactory information is encoded with numerous types of chemical substances, called odorants (Polak & Baliguet 1978). Odorants are captured by olfactory receptor neurons in the olfactory (nasal) epithelium, and those neurons that express the same odorant receptor genes project to specific glomeruli in the olfactory bulb, situated in most rostral region of the telencephalon (reviewed in Axel 2005). In the olfactory bulb, several types of projection neurons and interneurons elaborate highly ordered neuronal circuits, which play essential roles in processing odorant information (reviewed in Greer 1991). Two types of projection neurons, mitral and tufted cells, receive olfactory receptor innervations, and extend axons to several regions in the telencephalon, such as the olfactory cortex, olfactory tubercle, anterior olfactory nucleus, entorhinal cortex and several nuclei of the amygdala, where the odor information is further processed or stored (Zou et al. 2001). Thus, the vertebrate olfactory system comprises several neuronal components of the peripheral and central nervous system, and each constituent plays pivotal roles in perception, transduction and integration of the olfactory information (reviewed in Axel 2005; Wilson & Mainen 2006; Fig. 1).

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Figure 1. Schematic illustration of the vertebrate olfactory system. The adult olfactory system consists of three major components; the olfactory epithelium (OE), olfactory bulb (OB) and olfactory cortex (OC). Olfactory receptor neurons (red arrows) reside in the OE and extend their axons (olfactory nerve) to the olfactory bulb. Mitral cells and tufted cells (blue arrows) receive input from the receptor neurons, and further project toward the OC, which occupy the ventral part of the telencephalon. LOT, lateral olfactory tract; NC, nasal cavity; Ncx, neocortex.

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Although accumulating data emphasize the physiological and functional mechanisms underlying odor detection and discrimination (Wilson & Mainen 2006; Schaefer & Margrie 2007), the genetic programs that regulate the development of the olfactory system are not well understood. The vertebrate olfactory system develops during the early embryonic stages, and each component is mutually influenced during development, making it difficult to dissect cellular and molecular mechanisms that are required for specific processes in the developing olfactory system.

The Pax6 gene encodes a transcription factor containing two types of DNA binding domains, paired box and homeobox (Stoykova & Gruss 1994; Callaerts et al. 1997). Pax6 has conserved roles in the developing sensory organs and brains across vertebrate and invertebrate species (reviewed in Osumi 2001; Simpson & Price 2002; van Heyningen & Williamson 2002). It has been reported that mouse and rat strains with compromised Pax6 function display severe abnormalities in the developing olfactory system; including dysgenesis of the olfactory epithelium (Matsuo et al. 1993; Davis & Reed 1996; Anchan et al. 1997) and mislocation or lacking specific neuronal subtypes in the olfactory bulb and olfactory cortex (Lopez-Mascaraque et al. 1998; Jimenez et al. 2000; Hirata et al. 2002; Nomura & Osumi 2004; Nomura et al. 2006). These lines of evidence indicate that Pax6 plays pivotal roles in distinct developmental processes of the olfactory system. Here we introduce previous and recent findings on the role of Pax6 in: (i) development of the olfactory placode/epithelium; (ii) positioning and axon guidance of the olfactory bulb neurons; (iii) migration and alignment of olfactory cortex neurons; and (iv) generation of specific interneuron subtypes in the postnatal olfactory bulb.

Role of Pax6 in the developing olfactory placode and epithelium

During early embryonic stages, Pax6 is expressed in the front-nasal region including the olfactory placode, which gives rise to the olfactory epithelium in later development (Matsuo et al. 1993; Grindley et al. 1995; Fig. 2A–D). In homozygous Pax6 mutant mice (Sey/Sey) and rats (rSey/rSey), development of the olfactory placode is severely compromised, which results in the absence of olfactory receptor neurons (Hill et al. 1991; Matsuo et al. 1993; Grindley et al. 1995; Davis & Reed 1996; Jimenez et al. 2000; Fig. 2E,F). Several studies have shown that the development of the olfactory placode is regulated by the interaction between epithelial and mesenchymal tissues in the front-nasal regions, and that the retinoid-mediated signaling pathway is an essential constituent of this interaction (Osumi-Yamashita et al. 1990; LaMantia et al. 1993, 2000; Bhasin et al. 2003). Retinoic acid (RA), a derivative of vitamin A, is produced by the front-nasal region and considered to be a key molecule to trigger the retinoid-dependent signaling (reviewed in Rawson & LaMantia 2006). Importantly, reporter gene expression responding to the RA-signaling is not detected in the front-nasal region of the Sey/Sey mutant (Anchan et al. 1997). Furthermore, the expression of an RA-generating enzyme such as Raldh-3 is completely absent from the surface ectoderm of the rSey2/rSey2 mutant (Suzuki et al. 2000), suggesting that impaired development of the nasal placode in the Pax6 mutant might be due to a lack or defect of the RA-dependent pathway.

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Figure 2. Abnormal development of the olfactory placode-derived tissues in the Pax6 mutant. (A, B) Whole mount in situ hybridization with Pax6 probe on E11.5 wild type rat embryo. Pax6 is expressed in the forebrain (FB), eye and spinal cord (Sp). (C, D) Immunostaining with anti-Pax6 antibody on E11.5 embryos showing Pax6 expression in the nasal placode (OP, arrowheads in D) and forebrain (FB). (E, F) Immunostaining with antitype III β-tubulin antibody (an early neuronal marker) in coronal sections of E12.5 wild type (WT, E) and Pax6 mutant (rSey2/rSey2, F) rat embryos. In the WT front-nasal region, the olfactory neurons are differentiated in the invaginated olfactory epithelium (OE) (arrowheads in E). In contrast, neither morphological invagination nor neuronal differentiation is discernable in the front-nasal regions of the Pax6 mutant (F). Arrows in E and F indicate neurons in the ventral telencephalon. Bars, 500 µm (A, B); 200 µm (C, D); 100 µm (E, F).

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Previous studies indicated that neural crest-derived cells (NCDCs) also play crucial roles in the development of the front-nasal tissue (Osumi-Yamashita et al. 1994, 1997). During early embryogenesis, NCDCs derived from the midbrain contribute to the mesenchymal components in the front-nasal mass (Osumi-Yamashita et al. 1994). Furthermore, impaired migration of the midbrain-derived NCDCs has been shown in Pax6 mutant mouse and rat embryos (Matsuo et al. 1993; Osumi-Yamashita et al. 1997; Kanakubo et al. 2006). In Pax6 homozygous embryos, the migrating NCDCs cannot invade into the front-nasal mass (Matsuo et al. 1993; Osumi-Yamashita et al. 1997). The abnormal migration is due to non-cell autonomous defects in the migrating NCDCs (Osumi-Yamashita et al. 1997). Recent studies also indicated that HNK-1 carbohydrate epitope and the GlcAT-P gene that encodes an enzyme for the synthesis of the HNK-1 epitope are ectopically expressed in the facial ectoderm of the Pax6 mutant rat (Nagase et al. 2001, 2003). Furthermore, the HNK-1 epitope has a potential to inhibit the NCDCs migration (Nagase et al. 2003), suggesting that the impaired migration of the NCDCs might be due to ectopic expression of HNK-1 epitope in the mutant, and that Pax6 plays responsible role in the normal development of the front-nasal region via regulating the expression of those molecules.

Role of Pax6 in the developing olfactory bulb

The olfactory bulb represents the protrusion from the rostral part of the telencephalon, and functions as the primary processing center of olfactory information (Lopez-Mascaraque & de Castro 2002). It consists of several types of neurons that have distinct features and functions (Hinds 1968; Pinching & Powell 1971). The mitral cells are projection neurons in the olfactory bulb, and are known to receive input from the olfactory epithelium. These cells also extend axons toward the olfactory cortex, thereby transducing the olfactory information to higher information-processing centers. Interestingly, in the Pax6 homozygous mutant, the olfactory bulb does not develop at the rostral part of the telencephalon. Instead, the olfactory bulb-like structure (OBLS) is formed at the lateral part of the telencephalon in the mutant (Lopez-Mascaraque et al. 1998; Jimenez et al. 2000; Hirata et al. 2002; Fig. 3A,B). The OBLS consists of the mitral cells, which extend axons to the olfactory cortex similar to normal embryos (Jimenez et al. 2000; Hirata et al. 2002; Fig. 3C,D), although the orientation of each cell is severely compromised (Lopez-Mascaraque et al. 2005).

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Figure 3. Mislocation of the olfactory bulb in the Pax6 mutant. (A, B) Immunostaining with anti-Neuropilin-1 (NP1) antibody in E14.5 wild type (WT, A) and Pax6 mutant rat (rSey2/rSey2, B) telencephalon. NP1 marks the mitral cells and their axonal bundles, known as the lateral olfactory tract (LOT). White and black arrows indicate the olfactory bulb in WT and the olfactory bulb-like structure in the Pax6 mutant, respectively. (C, D) In situ hybridization with neuropilin-1 (np1) probe in the WT olfactory bulb (C) and the rSey2/rSey2 mutant olfactory bulb-like structure (OBLS) sections (D). mcl, mitral cell layer. Bars, 1 mm (A, B); 50 µm (C, D).

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Since the mitral cells are the first differentiating neurons in the olfactory bulb (Hinds 1968; Bayer 1983), mislocation of mitral cells seems to be a primary defect in the abnormal development of the olfactory bulb in the Pax6 mutant. To explore how Pax6 regulates the olfactory bulb formation, we examined the origins and migratory routes of mitral cells by using a whole-embryo culture system. Cell tracing analysis indicated that mitral cells are born at the rostral part of the telencephalon in both the wild type (WT) and Pax6 mutant. In the latter, however, mitral cells misrouted to the lateral part of the mutant telencephalon (Nomura & Osumi 2004). Furthermore, cell transplantation experiments revealed that the abnormal migration of the mitral cells is due to non-cell autonomous defects in the migrating cells. Previous studies suggested that projection of the olfactory receptor neurons to the telencephalon (olfactory nerve) is required for induction of the olfactory bulb (Stout & Graziadei 1980; Graziadei & Monti-Graziadei 1992). In the Pax6 homozygous mutant, olfactory nerve projections are totally absent (Jimenez et al. 2000). Therefore, it is possible that mislocation of the mitral cells is a secondary defect due to the loss of olfactory nerve innervations in the mutant. To address this possibility, we eliminated the influences of olfactory innervations by surgical ablation of the olfactory epithelium in WT embryos, prior to olfactory nerve projection. Despite the absence of olfactory nerve innervations, the mitral cells were still positioned at the rostral part of the telencephalon, suggesting that misrouting of the mitral cells in the Pax6 mutant is independent to the loss of olfactory innervations (Nomura & Osumi 2004). Concomitantly, overexpression of Pax6 expression vector into the Pax6 mutant telencephalon restored the mislocation of the mitral cells, indicating that Pax6 functions in the telencephalon for normal olfactory bulb development. These results indicate that Pax6 regulates the positioning of the olfactory bulb by restricting the migration of the mitral cells at the rostral part of the telencephalon (Nomura & Osumi 2004).

What are the molecules downstream of Pax6 that control the development of the olfactory bulb? Several studies have shown that FGF signaling has essential roles in the developing olfactory bulb. Mice lacking fgfr1 function or compromising fgf8 expression have a hypoplastic olfactory bulb (Garel et al. 2003; Hebert et al. 2003). To investigate whether fgf signaling is also altered in the Pax6 homozygous mutant, we examined the expression patterns of an FGF ligand and receptors in the telencephalon of Pax6 mutant. In situ hybridization identified the robust expression of fgf8 in the rostral midline of the mutant telencephalon, as in the case of WT (Fig. 4A,B). In contrast, marked reduction in the expression level of fgfr1 and fgfr3 were evident in the Pax6 mutant telencephalon, compared with the WT (Fig. 4C–F). A previous study suggested that FGF signaling is required for olfactory bulb protrusion, through the regulation of local cell proliferation (Hebert et al. 2003). Our results imply that FGF-signaling is also impaired in the Pax6 mutant telencephalon, which could explain the lacking protrusion of the mutant olfactory bulb.

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Figure 4. Low expression of FGF receptors in the Pax6 mutant. (A, B) In situ hybridization with fgf8 probe in E12.5 WT and rSey2/rSey2 telencephalon. fgf8 expression was not altered in the rSey2/rSey2 telencephalon, compared with that in the wild type (WT). (C–F) In situ hybridization with fgfr1 (C, D) and fgfr3 probes (E, F) in E12.5 WT (C, E) and rSey2/rSey2 (D, F) telencephalon. Note the expression of fgf receptors in the developing telencephalon with a caudal-high to rostral-low gradient. Compared with WT, the expression levels of fgfr1 and fgfr3 are severely reduced in the rSey2/rSey2 telencephalon. The experimental procedure of in situ hybridization was described previously (Osumi-Yamashita et al. 1997). Bars, 100 µm (A–F).

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Role of Pax6 in the developing olfactory cortex

The olfactory cortex functions as a secondary processing center of odor information, in which distinct types of neurons constitute a three-layered laminar structure (Bayer 1986; Valverde & Santacana 1994). Previous studies indicated that the majority of olfactory cortex neurons are derived from the ventral pallium, in which Pax6 is strongly expressed during embryogenesis, and that the Pax6 mutant lacks subpopulation of these neurons (de Carlos et al. 1996; Yun et al. 2001; Hirata et al. 2002). We also reported that neurons in the superficial layer of the olfactory cortex are deficient in the Pax6 mutant (Nomura et al. 2006). These neurons originate from the dorsal part of the telencephalon, and migrate ventrally, stopping migration at the pallial/subpallial boundary (PSB), which corresponds to the presumptive olfactory cortex (Fig. 5A). In the Pax6 mutant, these neurons migrate ventrally, but ignore the PSB and invade the ventral telencephalon (Fig. 5B). Cell transplantation experiments revealed that this abnormality is due to non-cell autonomous defects in the migrating cells. By screening gene(s) that are responsible for neuronal migration, we identified ephrin-A5 as a candidate molecule that regulates the migration of the olfactory cortex neurons. ephrin-A5 is expressed at the ventral part of the telencephalon (Fig. 5C), and expression level is severely decreased in the Pax6 mutant. Mis-expression of ephrin-A5 expression vector into the ventral part of the mutant telencephalon restores the migratory defects of the olfactory cortex neurons. Furthermore, ephrin-A5 deficient mice display a phenotype similar to that of the Pax6 mutant: the olfactory cortex neurons do not stop at the PSB, and invade the ventral part of the telencephalon. These results indicate that Pax6 regulates the migration of the olfactory cortex neurons by regulating ephrin-A5 expression. Gain- and loss-of-function studies have revealed that ephrin-A5 has a repulsive activity for migrating olfactory cortex neurons, thereby preventing invasion of the neurons into the ventral telencephalon (Nomura et al. 2006; Fig. 5D).

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Figure 5. Migratory defects of olfactory cortex neurons in the Pax6 mutants. (A, B) Comparison of migration patterns of green fluorescent protein (GFP)-labeled olfactory cortex neurons between wild type (WT) (A) and rSey2/rSey2 (B) embryos. In the WT, GFP-positive neurons align at the pallium/subpallium boundary (white arrows in A). In contrast, in the rSey2/rSey2 mutant, GFP-positive neurons invade the ventral part of the telencephalon (arrowheads in B). (C) The expression pattern of ephrin-A5 in E12.5 WT rat telencephalon. An arrow indicates the pallium/subpallium boundary. (D) Schematic illustration of migration patterns of the olfactory cortex neurons. In the WT, olfactory cortex neurons migrate ventrally (red arrows) and align at the presumptive olfactory cortex (OC), with respect to the border of ephrin-A5 expression (brown-shaded area). In contrast, these neurons do not align at the Pax6 mutant olfactory cortex, due to the lack of ephrin-A5 expression in the ventral part of the telencephalon. Bar, 100 µm (A–C).

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It has been shown that the projection patterns of mitral cell axons in the olfactory cortex are also affected in the Pax6 mutant (Hirata et al. 2002). Mitral cells extend axons on the surface of the olfactory cortex, which fasciculate to form a tight axon bundle called the lateral olfactory tract (LOT). During later embryogenesis, mitral cell axons develop collateral branches, which extend and project to the caudal part of the olfactory cortex (Hirata & Fujisawa 1999). In the Pax6 mutant, however, some axons in the LOT de-fasciculate and ectopically extend in the olfactory cortex (Hirata et al. 2002). Furthermore, collateral formation of the mitral cells is also severely compromised in the Pax6 mutant, and thus only a few axonal branches occupy the olfactory cortex (Hirata et al. 2002). It has been shown that a specific neuronal population known as ‘lot cells’ is involved in LOT development (Sato et al. 1998). During early embryonic stages, lot cells are born at the dorsal part of the telencephalon, and migrate ventrally, then populate at the presumptive LOT region, similar to the olfactory cortex neurons (Tomioka et al. 2000; Kawasaki et al. 2006). Curiously, the distribution of the lot cells are not altered in the Pax6 mutant olfactory cortex (Hirata et al. 2002), suggesting that LOT defects in the mutant are independent of the lot cells. Interestingly, collateral formation of the mammlio-tegmental tract is also compromised in the Pax6 mutant (Valverde et al. 2000), suggesting that Pax6 regulates common mechanism(s) underlying collateral axonal branching in the developing brain.

Role of Pax6 in the generation of olfactory bulb interneurons

The development of the olfactory system continues after birth, and in some regions new neurons are persistently generated during adult life (Kempermann 2006). The generation of olfactory bulb interneurons is particularly investigated as a model of postnatal/adult neurogenesis (Temple 2001; Alvarez-Buylla & Garcia-Verdugo 2002). Interneurons are functionally essential constituents of the olfactory bulb, and they are subdivided into two major subtypes: granule cells (GCs) and periglomerular cells (PGCs), which are discriminated based on their morphological, physiological and chemical characteristics (Kosaka et al. 1995; Lledo et al. 2004). GCs are homogeneously GABAergic, whereas PGCs are a heterogeneous population including GABAergic and dopaminergic neurons. These neurons are supplied from neural stem cells that reside in the telencephalic subventricular zone, and migrate tangentially toward the olfactory bulb via the ‘rostral migratory stream (RMS)’. It was previously demonstrated that Pax6 is expressed in neuronal progenitors in the RMS, and plays an important role in subtype specification of olfactory interneurons (Hack et al. 2005; Kohwi et al. 2005). Overexpression of Pax6 in the adult neuronal progenitors in the RMS increases the number of dopaminergic PGCs (Hack et al. 2005). Transplantation studies of the neuronal progenitor cells also revealed the cell-autonomous requirements of Pax6 in differentiation of the dopaminergic PGCs (Kohwi et al. 2005). Furthermore, the number of dopaminergic PGCs is decreased in Pax6 heterozygous mutant mice (Dellovade et al. 1998; Kohwi et al. 2005). We recently identified that the number of other PGC subtypes is also reduced in the olfactory bulb of Pax6 heterozygous mutant (H. Haba, T. Nomura and N. Osumi unpubl. data, 2007), suggesting that a proper dose of Pax6 is essential for differentiation and/or maintenance of these neurons.

A variety of transcription factors such as Dlx-1/–2 (Anderson et al. 1997), Gsh-1/–2 (Toresson & Campbell 2001), Arx (Yoshihara et al. 2005), and Sp8 (Waclaw et al. 2006) are differentially expressed in the olfactory interneurons, and combined expression of these genes seems to establish ‘transcriptional codes’ for specification of interneurons (Allen et al. 2007). Moreover, several extracellular proteins and their receptors such as slits (Wu et al. 1999; Nguyen-Ba-Charvet et al. 2004; Sawamoto et al. 2006), neuregulins (Ghashghaei et al. 2006), and Reelin (Hack et al. 2002) are involved in proper migration and differentiation of olfactory bulb interneurons. Exploring the genetic interactions between these molecules and Pax6 is thus essential to understand the control system that regulates the interneuron development.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Conclusions
  5. Acknowledgments
  6. References

Although recent advances in developmental neurobiology have unveiled genetic programs that govern the brain formation, several aspects of the highly orchestrated processes of the olfactory system development remain to be deciphered. In addition to molecular embryological approaches, spatial and temporal manipulations of the functions of specific genes are needed to explore various cellular and molecular interactions. Future analysis dissecting Pax6 function in a cell and tissue-specific manner will not only provide a better understanding of the role of this transcription factor in the developing olfactory system, but also shed light on the molecular mechanisms underlying the developmental processes of the vertebrate sensory system.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Conclusions
  5. Acknowledgments
  6. References

This work was supported by Grants-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports and Technology (18053003 to T. N.), and by the Core Research for Evolutional Science and Technology from Japan Science and Technology Agency (to N. O.). We thank Drs Nobuyuki Ito for providing mouse fgfr1, fgfr2, fgfr3, and fgfr4 cDNAs, Koji Tamura for providing mouse fgf8 cDNA, Masanori Takahashi for helpful comments on the manuscript, and Keiko Numayama-Tsuruta for providing whole mount in situ hybridization images of Pax6 and critical reading on the manuscript. We also thank Ms Noriko Takashima, Nao Kamata, Michi Otonari, Hisako Yusa, Ayumi Ogasawara, Sayaka Makino, Yumi Watanabe and Makiko Hoshino for animal care and technical assistance.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Conclusions
  5. Acknowledgments
  6. References