O.R. is an incumbent of the Bernstein−Mason Professorial Chair of Neurochemistry. Our research has been supported in part by the Israel Science Foundation (grants no. 47/10 and 322/13), Jerome Lejeune Foundation, Minerva foundation with funding from the Federal German Ministry for Education and Research, Helen and Martin Kimmel Institute for Stem Cell Research, the Benoziyo Center for Neurological diseases, the Kekst Family Center for Medical Genetics and the David and Fela Shapell Family Center for Genetic Disorders Research. The work has been supported by the Deutsche Forschungsgemeinschaft (SFB 1080 to RN and JV) and by the European Research Council (ERC-AG "LiPsyD" to RN).

#### **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 30 November 2014; accepted: 06 February 2015; published online: 04 March 2015.*

*Citation: Greenman R, Gorelik A, Sapir T, Baumgart J, Zamor V, Segal-Salto M, Levin-Zaidman S, Aidinis V, Aoki J, Nitsch R, Vogt J and Reiner O (2015) Noncell autonomous and non-catalytic activities of ATX in the developing brain. Front. Neurosci. 9:53. doi: 10.3389/fnins.2015.00053*

*This article was submitted to Neurogenesis, a section of the journal Frontiers in Neuroscience.*

*Copyright © 2015 Greenman, Gorelik, Sapir, Baumgart, Zamor, Segal-Salto, Levin-Zaidman, Aidinis, Aoki, Nitsch, Vogt and Reiner. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Proteoglycans and neuronal migration in the cerebral cortex during development and disease

#### Nobuaki Maeda\*

*Neural Network Project, Department of Brain Development and Neural Regeneration, Tokyo Metropolitan Institute of Medical Science, Setagaya, Japan*

Chondroitin sulfate proteoglycans and heparan sulfate proteoglycans are major constituents of the extracellular matrix and the cell surface in the brain. Proteoglycans bind with many proteins including growth factors, chemokines, axon guidance molecules, and cell adhesion molecules through both the glycosaminoglycan and the core protein portions. The functions of proteoglycans are flexibly regulated due to the structural variability of glycosaminoglycans, which are generated by multiple glycosaminoglycan synthesis and modifying enzymes. Neuronal cell surface proteoglycans such as PTPζ , neuroglycan C and syndecan-3 function as direct receptors for heparin-binding growth factors that induce neuronal migration. The lectican family, secreted chondroitin sulfate proteoglycans, forms large aggregates with hyaluronic acid and tenascins, in which many signaling molecules and enzymes including matrix proteases are preserved. In the developing cerebrum, secreted chondroitin sulfate proteoglycans such as neurocan, versican and phosphacan are richly expressed in the areas that are strategically important for neuronal migration such as the striatum, marginal zone, subplate and subventricular zone in the neocortex. These proteoglycans may anchor various attractive and/or repulsive cues, regulating the migration routes of inhibitory neurons. Recent studies demonstrated that the genes encoding proteoglycan core proteins and glycosaminoglycan synthesis and modifying enzymes are associated with various psychiatric and intellectual disorders, which may be related to the defects of neuronal migration.

#### Edited by:

*Angelique Bordey, Yale University School of Medicine, USA*

#### Reviewed by:

*Andreas Faissner, Ruhr-University Bochum, Germany Kosei Takeuchi, Aichi Medical University, Japan*

#### \*Correspondence:

*Nobuaki Maeda, Department of Brain Development and Neural Regeneration, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya, Tokyo 156-8506, Japan<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Neurogenesis, a section of the journal Frontiers in Neuroscience*

> Received: *26 December 2014* Accepted: *07 March 2015* Published: *23 March 2015*

#### Citation:

*Maeda N (2015) Proteoglycans and neuronal migration in the cerebral cortex during development and disease. Front.

# Introduction

The extracellular matrix (ECM) is a complex network of molecules composed of proteoglycans, hyaluronic acid, fibrous proteins, and various glycoproteins, which fills up the extracellular space within all tissues and organs (Mouw et al., 2014). The ECM also retains water and ions, and constitutes the direct environment surrounding cells, in which multiple types of molecules are cross-linked to each other through protein-protein and protein-carbohydrate interactions, forming the three-dimensional meshworks (**Figure 1**). The ECM serves not only as a physical scaffold for tissue construction, but also as a dynamic field of signaling that regulates the behavior of cells. In the meshwork of ECM, various signal molecules such as growth factors and chemokines are stored, and the concentration gradients of morphogens such as BMPs and Wnts are also formed. The ECM also serves as an adhesive substrate for the cells, regulating their motility and shape. The structures of ECM are not fixed and static, but are dynamically reorganized by the biosynthesis of its components and their degradation by various proteases and glycanases. Thus, the dynamics of the ECM is quite important in the regulation of cell growth, differentiation, migration, adhesion and tissue morphogenesis.

Unlike other organs, the brain does not normally contain fibrillar collagens except for the basal lamina surrounding the blood vessels and surface of the brain. Instead, major components of brain ECM are proteoglycans. Until recently, many neuroscientists had believed that the brain contains almost no ECM, in spite of the early biochemical and histochemical pioneering work by Margolis et al. (1975, 1976) and Nakanishi (1983), showing the presence of a large amount of glycosaminoglycans and proteoglycans in the developing brain. In the early 1990s, the brain-specific chondroitin sulfate proteoglycans were biochemically characterized (Rauch et al., 1991; Maeda et al., 1992; Oohira et al., 1994), and then their cDNAs were cloned (Rauch et al., 1992; Maeda et al., 1994; Maurel et al., 1994; Watanabe et al., 1995). Then, many investigations revealed the importance of proteoglycans and ECM in the development and disorders of the brain (Franco and Muller, 2011; Maeda et al., 2011; Berretta, 2012; Soleman et al., 2013). However, even now, it seems that the significance of ECM molecules is underestimated in the field of neuroscience, except for reelin. It is desirable that more and more neuroscientists pay attention to the brain ECM molecules.

In this review, I will introduce the structure, binding partners and assembly of proteoglycans and glycosaminoglycans, and discuss their roles in the neuronal migration in the cerebral cortex and their emerging significance in human intellectual disability and psychiatric disorders.

# Assembly of Extracellular Matrix Components

In the developing cerebral cortex, very high levels of ECM molecules are expressed, the major components of which are

store various proteins such as chemokines, growth factors and axon guidance molecules. The ECM proteoglycans may bind with a CS/HS receptor on the cell surface such as RPTPσ. Cell surface proteoglycans may function as receptors or co-receptors for growth factors.

chondroitin sulfate proteoglycans. Neurocan, versican, aggrecan, and brevican are lectican family chondroitin sulfate proteoglycans expressed in the brain. Lecticans are secreted proteoglycans that bind with hyaluronic acid through the N-terminal G1 domain that contains an immunoglobulin-like loop and two link modules (**Figure 2**). These proteoglycans also have a C-type lectin domain at the C-terminal, and chondroitin sulfate chains are covalently attached to the region between the N- and C-terminal domains. The C-type lectin domain of lecticans binds with tenascin family proteins by protein-protein interaction independent of the carbohydrate moiety (Aspberg et al., 1997). Tenascin family proteins are oligomeric glycoproteins with EGF-like repeats and fibronectin-III domains. The brain contains hexameric tenascin-C and trimeric tenascin-R, which promote the assembly of lecticans by protein-protein interaction. On the other hand, hyaluronic acid is a very long polysaccharide consisting of repeating disaccharides of glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc), which are polymerized at the plasma membrane by hyaluronan synthases (HASs). Hyaluronic acids are anchored in the plasma membrane through HASs, or bound to hyaluronan receptors on the cell surface, such as CD44 and RHAMM (**Figure 1**). The tenascin-lectican complexes bind to the hyaluronic acids through the G1 domains of lecticans, which is stabilized by link proteins (Haplns), forming huge aggregates surrounding cells (**Figure 1**). It is considered that these huge aggregates serve as a basic framework to construct the ECM in the brain.

Besides secreted chondroitin sulfate proteoglycans, there are also cell surface proteoglycans. PTPζ /RPTPβ and neuroglycan C are major cell surface chondroitin sulfate proteoglycans with a membrane-spanning region. PTPζ is a receptor-type protein tyrosine phosphatase that is synthesized as a chondroitin sulfate proteoglycan (Krueger and Saito, 1992; Maeda et al., 1994). PTPζ has an N-terminal carbonic anhydrase-like domain, a fibronectin-III domain, a membrane-spanning region and two C-terminal tyrosine phosphatase domains. The extracellular domain of this receptor generated by alternative splicing is secreted as a major soluble chondroitin sulfate proteoglycan in the developing brain, phosphacan (Maurel et al., 1994). Phosphacan binds with multiple proteins including pleiotrophin, midkine, tenascins, contactin, and NCAM (Peles et al., 1998). Neuroglycan C is a transmembrane chondroitin sulfate proteoglycan with an EGF module at the juxtamembrane region of the extracellular domain (Watanabe et al., 1995). Chondroitin sulfate-modification of neuroglycan C is developmentally and regionally regulated, and the expression of the non-proteoglycan form increases with development (Aono et al., 2004).

Another major group of proteoglycans in the developing nervous system is heparan sulfate proteoglycans: syndecans and glypicans (**Figure 2**). The syndecan family is composed of four members, syndecan-1 to -4, each of which has an extracellular domain, a transmembrane region and a conserved short C-terminal cytoplasmic domain (Lambaerts et al., 2009). The N-terminal portion of the extracellular domain of syndecans is

modified with heparan sulfate chains. The extracellular domains of syndecan-1 and -4 may be additionally decorated with chondroitin sulfate chains near the transmembrane region (Deepa et al., 2004). It has been considered that most of the extracellular ligand molecules interact with syndecans through binding with heparan sulfate portions. The transmembrane domains of syndecans play important roles in their ligand-induced multimerization and the subsequent signaling (Choi et al., 2005). The intracellular domains of syndecans are divided into two conserved regions (C1 and C2) and a variable region (V), which interact with various kinases and intracellular cytoplasmic components such as src family kinases, CASK, and syntenin (Lambaerts et al., 2009).

Glypicans are glycosyl-phosphatidylinositol (GPI)-anchored heparan sulfate proteoglycans, composed of six family members (glypican-1 to -6) carrying 2–5 heparan sulfate chains (Filmus et al., 2008; Filmus and Capurro, 2014). Glypican-5 was reported also to be modified with chondroitin sulfate in rhabdomyosarcoma cells (Li et al., 2011). The core proteins of glypicans consist of an α-helical domain containing 14 evolutionarily conserved Cys residues, a heparan sulfate-attachment region near the C-terminus, and the C-terminal GPI-anchor attachment signal sequence. Biochemical and genetic studies demonstrated that glypicans bind and regulate Hedgehogs, Wnt, bone morphogenetic proteins and fibroblast growth factors (FGFs) (Filmus and Capurro, 2014). In particular, genetic studies using Drosophila demonstrated that glypicans (Dally and Dally-like) play critical roles in the gradient formation of morphogens during wing development (Wu et al., 2010; Raftery and Umulis, 2012). Although it has long been believed that the core proteins of glypicans adopt a globular shape, a recent X-ray crystallographic study indicated that the structure of glypican-1 is actually cylindrical (Svensson et al., 2012).

Testicans are extracellular chondroitin/heparan sulfate proteoglycans, which have been poorly characterized to date. They are composed of three family members (testican-1 to -3), characterized by an N-terminal testican-specific domain, a follistatin-like domain, an extracellular calciumbinding domain, a thyroglobulin-like domain, and a domain with glycosaminoglycan-attachment sites (Schnepp et al., 2005).

# Structure and Biosynthesis of Chondroitin Sulfate and Heparan Sulfate

Chondroitin sulfate and heparan sulfate are unbranched sulfated polysaccharides covalently attached to the serine residues in proteoglycan core proteins via common linkage tetrasaccharides, GlcAβ1-3galactose(Gal)β1-3Galβ1-4xylose(Xyl)β1-O-Ser (**Figure 3**). Chondroitin sulfate is composed of repeating disaccharide units of N-acetylgalactosamine (GalNAc) and GlcA, (GlcAβ1-3GalNAcβ1-4)n, whereas heparan sulfate is composed of repeating disaccharide units of GlcNAc and GlcA, (GlcAβ1- 4GlcNAcα1-4)n. Biosynthesis of these polysaccharides is initiated by the addition of Xyl residues to the specific serine residues in

core proteins by xylosyl transferases (XYLT1, 2) (Nadanaka and Kitagawa, 2008; Mikami and Kitagawa, 2013; Mizumoto et al., 2013a). This is followed by the addition of two Gal residues and a GlcA residue by galactosyltransferase-I (B4GALT7), galactosyltransferase-II (B3GALT6) and glucuronyltransferase-I (B3GAT3), respectively. After that, repeating disaccharide units of chondroitin or heparan sulfate are polymerized in the Golgi apparatus. Chondroitin sulfates are polymerized by chondroitin sulfate synthases (CHSY-1 to -3), chondroitin polymerization factor (CHPF) and chondroitin sulfate N-acetylgalactosaminyltransferases (CSGalNAcT-1, -2), whereas heparan sulfates are polymerized by EXT family members (EXT1, EXT2, EXTL1, EXTL2, EXTL3). Since these glycosaminoglycans use a common linkage tetrasaccharide, the chain initiation step determines whether a chondroitin or a heparan chain elongates. If the first GalNAc residue is added to the linkage tetrasaccharide possibly by CSGalNAcT-1 or -2, a chondroitin chain is elongated by the CHSY/CHPF complex. If a GlcNAc residue is alternatively added to the linkage tetrasaccharide possibly by EXTL 2 or 3, a heparan chain is polymerized by the EXT1/EXT2 complex. Although the genes encoding these biosynthetic enzymes have been identified, almost nothing is known about the mechanism for the selection of glycosaminoglycan types at the chain initiation step.

After the polymerization of repeating disaccharides, they are heavily modified by the C5 epimerization of GlcA residues and sulfation reactions (Nadanaka and Kitagawa, 2008; Mikami and Kitagawa, 2013; Mizumoto et al., 2013a) (**Figure 3**). The majority of the GalNAc residues in chondroitin sulfate are 4-O-sulfated by chondroitin 4-O-sulfotransferases (C4ST-1 to - 3) or 6-O-sulfated by 6-O-sulfotransferases (C6ST-1 and -2). Although many of the disaccharide units in chondroitin sulfate are mono-sulfated A units [GlcAβ1-3GalNAc(4-SO4)] or C units [GlcAβ1-3GalNAc(6-SO4)], they may be further sulfated by GalNAc 4-sulfate 6-O-sulfotransferase (GalNAc 4S-6ST) or chondroitin uronyl 2-O-sulfotransferase (UST), generating di-sulfated disaccharides, E units [GlcAβ1-3GalNAc(4, 6-bis-SO4)] or D units [GlcA(2-SO4)β1-3GalNAc(6-SO4)], respectively. Furthermore, some of the GlcA residues are C5-epimerized to iduronic acid (IdoA) by dermatan sulfate epimerases 1 and 2 (encoded by DSE and DSEL, respectively). The resulting IdoAα1-3GalNAc units are sulfated by dermatan 4-O-sulfotransferase (D4ST), generating an iA unit [IdoAα1-3GalNAc(4-SO4)], which may be further sulfated by UST, generating an iB unit [IdoA(2-SO4)α1- 3GalNAc(4-SO4)]. Chondroitin sulfate with a high content of IdoA is often called dermatan sulfate.

The modification of heparan sulfate begins with the N-sulfation reaction by N-deacetylase/N-sulfotransferase (NDST-1 to -4), which removes acetyl groups from some of the GlcNAc residues in the heparan sulfate chain and replaces them with sulfate groups (**Figure 3**). Then, some of the GlcA residues are C5 epimerized to IdoA by heparan sulfate glucuronyl C5-epimerase (HS C5-EP), followed by O-sulfation reactions. The O-sulfation includes 2-O-sulfation of GlcA/IdoA residues by heparan sulfate 2-O-sulfotransferase (H2ST), 3- O-sulfation of glucosamine (GlcN) units by heparan sulfate 3-O-sulfotransferases (H3ST-1, -2, -3A, -3B, -4, -5, and -6), and 6-O-sulfation of GlcN units by heparan sulfate 6-Osulfotransferases (H6ST-1 to -3). Because N-sulfation of GlcN units by NDSTs generates substrates for the subsequent modification enzymes, highly N-sulfated regions in heparan sulfate are also highly modified by C5-EP and various O-sulfotransferases. Thus, heparan sulfate chains display domain structures: highly modified NS-domains, poorly modified NA-domains characterized by stretches of N-acetylated disaccharide units, and the interspacing NA/NS-domains composed of both N-acetylated and N-sulfated disaccharide units.

# Binding Partners of Glycosaminoglycans

Recent studies using microarrays and surface plasmon resonance revealed that chondroitin sulfate and heparan sulfate chains bind with many proteins that play important roles in brain development, especially neuronal migration (Deepa et al., 2002; Kawashima et al., 2002; Maeda et al., 2006; Shipp and Hsieh-Wilson, 2007; Conrad et al., 2010; Rogers et al., 2011; Mizumoto et al., 2013b). Both heparan sulfate and chondroitin sulfate chains bind with various axon guidance molecules in a sulfation patterndependent manner (Shipp and Hsieh-Wilson, 2007). While Slit2 shows a preference for heparan sulfate sequences that contain 6-O-sulfation and N-sulfation, netrin 1 requires sulfation at the 2-O-, 6-O-, and N-positions. Semaphorin5B (Sema5B), ephrinA1 and ephrinA5 prefer 2-O- and N-sulfation. On the other hand, all of these axon guidance molecules bind strongly with E unit-rich highly sulfated chondroitin sulfate E from squid cartilage (CS-E) (Shipp and Hsieh-Wilson, 2007). Sema5B also binds moderately with D unit-rich shark cartilage chondroitin sulfate D (CS-D), and weakly with A unit-rich whale cartilage chondroitin sulfate A (CS-A), pig skin dermatan sulfate (CS-B) and C unit-rich shark cartilage chondroitin sulfate C (CS-C). EphrinA1 binds moderately with CS-C, and weakly with CS-B and CS-D. Slit2, netrin1 and ephrinA5 bind only weakly with CS-A, -C, -D, and -B. In addition, it has been reported that Sema3A binds strongly with CS-E (Dick et al., 2013). EphrinA3 also binds with heparan sulfate and chondroitin sulfate, although the structural requirement is unknown (Irie et al., 2008; Conrad et al., 2010).

Neurotrophin family growth factors [nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4)] bind strongly with CS-E (Rogers et al., 2011). BDNF, NT-3, and NT-4, but not NGF bind moderately with CS-A, whereas CS-C shows almost no binding to these proteins. PC12 cells express E unitrich chondroitin sulfate, and its removal upon chondroitinase ABC treatment significantly attenuated TrkA activation by NGF or NT-4, suggesting that endogenous chondroitin sulfate plays important roles in neurotrophin signaling (Rogers et al., 2011). Glial cell line-derived neurotrophic factor (GDNF) associates with heparan sulfate in a 2-O-sulfation-dependent manner, promoting the binding of this protein to its receptor component GFRα1 (Rickard et al., 2003).

Chemokines are a family of small proteins that induce chemotaxis of various cells including cortical interneurons (Marin, 2013). Glycosaminoglycans interact with chemokines such as CCL2 (MCP-1), CCL5 (RANTES), and CXCL12 (SDF-1) in a chain length- and sulfation pattern-dependent manner (Kuschert et al., 1999; Hirose et al., 2001). Among the chemokines, CXCL12 plays important roles in the tangential migration of cortical interneurons (see below). CXCL12 binds strongly with CS-E and the highly sulfated S domains in heparan sulfate, and also interacts weakly with CS-A, -B, -C, and -D (Murphy et al., 2004; Mizumoto et al., 2013b). In addition, it was revealed that versican interacts with CXCL12 through chondroitin sulfate chains (Hirose et al., 2001). It has been considered that these interactions in the ECM and cell surface contribute to the formation of immobilized or haptotactic gradients of chemokines (Kuschert et al., 1999).

Pleiotrophin and midkine are a family of multifunctional heparin-binding growth factors that bind with both heparan sulfate and chondroitin sulfate (Perez-Pinera et al., 2007; Muramatsu, 2014). These growth factors bind strongly with highly sulfated heparan sulfate and CS-E, moderately to CS-B and CS-D, and very weakly to CS-A (Maeda et al., 2003; Zou et al., 2003; Mizumoto et al., 2013b).

The structures of glycosaminoglycans are determined at least partly by the combinatorial expression of their modifying enzymes. During development of the brain, the sulfotransferases involved in the chondroitin/heparan sulfate synthesis show dynamic spatiotemporal expression patterns (Yabe et al., 2005; Mitsunaga et al., 2006; Ishii and Maeda, 2008b). This suggests that the expression of specific functional domains in these glycosaminoglycan chains is strictly regulated in the developing brain. Conway et al. (2011) showed that the expressions of HS2ST and HS6ST-1 are distinctively regulated at the optic chiasm, and the mutant mice lacking these genes exhibit different axon guidance defects. Interestingly, HS2ST−/<sup>−</sup> and HS6ST-1−/<sup>−</sup> phenotypes closely match those of Slit1−/<sup>−</sup> and Slit2−/−, respectively, suggesting that slit family proteins are regulated by specific sulfation of heparan sulfate.

# Ligand Binding to Proteoglycans

As described above, glycosaminoglycans bind with various protein ligands in a structure-dependent manner. However, the ligand binding to proteoglycans is extremely complex because many proteoglycans carry multiple glycosaminoglycan chains that may function cooperatively. Furthermore, cooperation between the core protein and attached glycosaminoglycan chains may also occur. Accordingly, proteoglycans usually exhibit much higher affinity and/or avidity for the ligand proteins than free glycosaminoglycans (Herndon et al., 1999). Thus, degradation of proteoglycan core proteins by extracellular proteases may terminate such cooperativity and release the ligand molecules, leading to the activation or inactivation of the signaling in a contextdependent manner. Cooperation is observed not only between glycosaminoglycans of the same type but also between heparan sulfate and chondroitin sulfate chains. Syndecan-1 and syndecan-4 carry both heparan sulfate and chondroitin sulfate chains, which cooperatively regulate the binding dynamics of pleiotrophin, midkine and FGF-2 to these proteoglycans (Deepa et al., 2004).

PTPζ /phosphacan binds to pleiotrophin and midkine, in which both chondroitin sulfate and core protein portions contribute to the interaction (Maeda et al., 1996, 1999, 2003). While intact phosphacan preparation shows low (Kd = 3 nM) and high affinity binding (Kd = 0.25 nM) for pleiotrophin, this proteoglycan exhibits only single very low affinity binding after chondroitinase ABC-treatment (Kd = 13 nM) (Maeda et al., 1996). This suggests that the binding affinity of phosphacan for pleiotrophin is regulated by the structural variation of chondroitin sulfate. In fact, the structure of chondroitin sulfate on phosphacan changes during rat brain development, and a slight increase in the content of oversulfated D unit drastically strengthens the binding of this proteoglycan to pleiotrophin (Maeda et al., 2003).

Another prominent example of the cooperation between the core protein and glycosaminoglycan chain in the signaling has been reported by the group of Filmus (Li et al., 2011; Filmus and Capurro, 2014). They revealed that glypican-3 and glypican-5 oppositely regulate the Hedgehog (Hh) signaling in rhabdomyosarcoma cell proliferation. Glypican-3 binds to Hh through its core protein, reducing the amount of Hh available to its receptor Patched 1 (Ptc1), with the consequent decrease in signaling. On the other hand, glypican-5 interacts with both Hh and Ptc1 through heparan sulfate and chondroitin sulfate chains, facilitating Hh-Ptc1 binding with the consequent increased signaling. The heparan sulfate chains of glypican-5 show a higher degree of sulfation than those of glypican-3, which may explain why the glycosaminoglycan chains of glypican-5 but not those of glypican-3 interact with Hh/Ptc1.

Sema5A is an axon guidance molecule that can exert both inhibitory and permissive effects on growing axons. Kantor et al. (2004) revealed that Sema5A interacts with the glycosaminoglycan portion of both chondroitin sulfate and heparan sulfate proteoglycans. The axonal heparan sulfate proteoglycans are required for the Sema5A-mediated attraction of growing axons of the fasciculus retroflexus. On the other hand, the extracellular chondroitin sulfate proteoglycans precisely localize Sema5A in a specific area, where Sema5A acts as a repulsive guidance cue for these growing axons. Thus, the bifunctional roles of Sema5A are regulated by chondroitin sulfate and heparan sulfate proteoglycans, demonstrating the cooperation among these two types of proteoglycans during the process of axon pathfinding.

Recently, Coles et al. (2011) reported that receptor protein tyrosine phosphatase σ (RPTPσ) is a receptor for both chondroitin sulfate and heparan sulfate proteoglycans. Heparan sulfate proteoglycans induce oligomerization of RPTPσ on the growth cone, leading to inactivation of the tyrosine phosphatase activity and growth promotion. On the other hand, extracellular chondroitin sulfate proteoglycans inhibit oligomerization of this receptor with consequent suppression of axon growth. It is considered that multiple RPTPσ molecules bind to the islands of high/intermediate sulfation on heparan sulfate chains (NS to NA/NS domains), which stabilize the receptor oligomers. Conversely, the receptor binding sites are considered to be sparsely distributed on chondroitin sulfate chains, and therefore receptor oligomerization cannot occur. If so, the signaling of RPTPσ should be highly dependent on the glycosaminoglycan structures. Proteoglycans bearing low sulfated heparan sulfate chains may Maeda Proteoglycans in brain

inhibit receptor oligomerization, and conversely proteoglycans bearing highly sulfated chondroitin sulfate may induce receptor oligomerization. It is also reported that another receptor tyrosine phosphatase, leukocyte common antigen-related phosphatase (LAR) is a functional receptor for chondroitin sulfate proteoglycan (Fisher et al., 2011).

Although the structure of glycosaminoglycans is basically determined by the biosynthetic processes, the sulfation pattern of heparan sulfate may be modified extracellularly by the endosulfatases, Sulf1 and Sulf2 (Nagamine et al., 2012). These sulfatases catalyze the desulfation of the 6-O-sulfate group from GlcN residues in the trisulfated disaccharides in heparan sulfate. Thus, highly sulfated NS domains in heparan sulfate are preferentially desulfated by these enzymes, leading to the change in the binding affinity of various ligands to heparan sulfate proteoglycans. This results in the activation or suppression of specific signaling molecules such as Wnt, GDNF, and FGF during various developmental processes (Ai et al., 2003, 2007; Wang et al., 2004). Thus, functions of proteoglycans are intricately regulated at multiple levels.

# Roles of Proteoglycans in Neuronal Migration

In the developing neocortex, postmitotic pyramidal neurons generated in the ventricular zone show a multipolar shape, and migrate in random directions in the subventricular and intermediate zones (**Figure 4**). After that, they transform into the bipolar shape and attach to the radial glial fibers, upon which they rapidly migrate toward the marginal zone. This multipolar-to-bipolar transition occurs when the neurons reach the subplate, suggesting that this layer contains critical factor(s) regulating neuronal behavior (Ohtaka-Maruyama et al., 2013). The radial migration of pyramidal neurons stops at the interface between the cortical plate and the marginal zone, forming the "inside-out" arrangement of neurons. On the other hand, inhibitory neurons tangentially migrate in the neocortex through the marginal zone, subplate, and lower intermediate/subventricular zones. These migration patterns of both excitatory and inhibitory neurons suggest that specific cortical layers play critical roles in the regulation of neuronal migration. Nakanishi (1983) demonstrated that glycosaminoglycans stained by colloidal iron distributed principally in the marginal zone and subplate in the developing mouse cerebral cortex. In the cortices of reeler mutants, where radial migration of pyramidal neurons is severely disturbed, most glycosaminoglycans are localized in the outer layer of the cortex. From such an expression pattern, it was suggested that glycosaminoglycans are involved in the neuronal migration and/or laminar pattern formation of the neocortex. Then, the brainspecific chondroitin sulfate proteoglycans were identified, and it was revealed that neurocan and phosphacan are richly expressed in the marginal zone and subplate (Oohira et al., 1994; Maeda et al., 1995; Meyer-Puttlitz et al., 1996) (**Figure 5**). Versican was also localized in these layers (Popp et al., 2003), raising the possibility that chondroitin sulfate proteoglycans regulate neuronal migration in the cortex.

As described above, PTPζ /phosphacan binds to pleiotrophin with high affinity (Maeda et al., 1996, 1999). Pleiotrophin induces oligomerization of PTPζ , which leads to the inactivation of its

tyrosine phosphatase activity, initiating downstream signaling (Meng et al., 2000; Fukada et al., 2006). Pleiotrophin is deposited along radial glial fibers, and PTPζ is expressed on the migrating pyramidal neurons, raising the possibility that pleiotrophin on radial glial fibers regulates the radial migration of excitatory neurons (Maeda and Noda, 1998). In fact, in vitro cell migration assay demonstrated that pleiotrophin-PTPζ signaling induces migration of cortical neurons (Maeda and Noda, 1998). Subsequently, this signaling system has been demonstrated to promote the migration of various types of normal and tumor cell in a chondroitin sulfate-dependent manner (Polykratis et al., 2005; Feng et al., 2010; Koutsioumpa et al., 2013). Phosphacan and free chondroitin sulfate suppress the pleiotrophin-induced neuronal migration by competitive inhibition of the binding between pleiotrophin and cell surface PTPζ (Maeda and Noda, 1998; Maeda et al., 1999). Soluble chondroitin sulfate proteoglycans such as phosphacan, neurocan and versican expressed in the subplate and marginal zone may regulate the migratory behavior of neurons by inhibiting pleiotrophin- PTPζ signaling.

Recently, it was found that neuroglycan-C is involved in the radial migration of pyramidal neurons from a study of the plant homeodomain (PHD) finger 6 (PHF6) gene (Zhang et al., 2013). PHF6 is an X-linked gene encoding the protein that has four nuclear localization signals and two PHD-type zinc finger domains, which functions as a transcription repressor. Mutations of PHF6 cause Börjeson-Forssman-Lehmann syndrome, characterized by intellectual disability associated with seizures, short stature, hypogonadism, hypometabolism, marked gynecomastia, truncal obesity, tapered fingers, narrow palpebral fissure, and large ears (Liu et al., 2014). Using in utero electroporation, Zhang et al. (2013) demonstrated that knockdown of PHF6 severely impaired the radial migration of cortical neurons. They also identified neuroglycan-C as a downstream target of PHF6. Knockdown of neuroglycan-C phenocopied the neuronal migration phenotype of PHF6 knockdown, suggesting that PHF6 controls the expression level of neuroglycan-C in the cortical neurons, thus regulating the radial neuronal migration. Neuroglycan C binds with pleiotrophin and midkine, in which its chondroitin sulfate portion increases the affinity of the core protein for these growth factors (Ichihara-Tanaka et al., 2006; Nakanishi et al., 2010). There is a possibility that pleiotrophin/midkineneuroglycan C signaling is involved in the radial migration in the cerebral cortex.

Pleiotrophin also binds with syndecan-3 in a heparan sulfatedependent manner (Raulo et al., 1994; Kinnunen et al., 1996). Like PTPζ , syndecan-3 is required for pleiotrophin-induced neuronal migration, suggesting that PTPζ and syndecan-3 are redundant pleiotrophin receptors on cortical neurons (Hienola et al., 2006). Syndecan-3 knockout mice showed delayed radial neuronal migration in the cortex, and this delay was partially caught up at ∼10 days after birth (Hienola et al., 2006). The binding of pleiotrophin to syndecan-3 triggers the phosphorylation of src, which then activates cortactin and modulates the assembly of the actin cytoskeleton. Syndecan-3 knockout mice also show migration defects of interneurons (Bespalov et al., 2011). At embryonic day 15, calbindin-positive precursors of interneurons were accumulated in the ganglionic eminence of syndecan-3 knockout mice, and the density of GABA-immunoreactive cells was lower in the dorsomedial cortex of adult knockout mice than in that of control mice. It has been suggested that GDNF binds to the heparan sulfate portion of syndecan-3 on interneurons, promoting their migration. On the other hand, syndecan-1 is expressed by the neural progenitor cells in the cerebral cortex (Wang et al., 2012). Knockdown of syndecan-1 using in utero electroporation resulted in the reduction of neural progenitor cells and the promotion of neuronal differentiation in the cortex (Wang et al., 2012). In these cortices, there were fewer cells in the ventricular/subventricular zone, and more neurons moved into the intermediate zone and cortical plate compared with the cortices electroporated with control plasmid. These findings suggest that syndecan-1 and syndecan-3 are differentially expressed and play distinct roles in the developing cortex.

# Roles of Glycosaminoglycans in Neuronal Polarization

During development of the mouse cerebrum, the disaccharide composition of chondroitin sulfate changes dynamically (Ishii and Maeda, 2008a). At embryonic day 16 (E16), the major components are A and C units, and then the content of A unit increases and that of C unit decreases until maturation. A small but significant amount of E unit is detected at E16 to 18, and the content decreases thereafter. D unit is a minor component, but is constantly detected during embryonic and postnatal development. Since D and E units contribute significantly to the binding of various ligand molecules as described above, we investigated the roles of these oversulfated structures in neuronal migration (Ishii and Maeda, 2008a). Using in utero electroporation, we introduced shRNA constructs for GalNAc 4S-6ST and UST into neural progenitor cells in the ventricular zone of the E14 cortex. At E18, the embryos were dissected out, and the migration of the cortical neurons was examined. Knockdown of both sulfotransferases severely disrupted the radial migration of cortical neurons. The neurons knocked down for these enzymes were accumulated in the subventricular zone and in the intermediate zone, and showed multipolar morphology. This suggested that oversulfated chondroitin sulfate is required for the multipolar-to-bipolar transition of pyramidal neurons.

Neuronal polarization of dissociated hippocampal pyramidal cells is a well-established in vitro model of multipolar-to-bipolar transition of newborn neurons (Dotti et al., 1988). Dissociated hippocampal pyramidal neurons extend several morphologically indistinguishable minor processes several hours after plating. Then, one of these minor processes extends rapidly and becomes an axon, and the other processes differentiate into dendrites. In contrast, the hippocampal neurons cultured in the presence of chondroitinase ABC extended multiple axon-like processes that were highly unstable and repeatedly extended and retracted (Nishimura et al., 2010). The morphology and behavior of the chondroitinase ABC-treated neurons were similar to those of multipolar neurons in the developing cortex. Furthermore, knockdown of GalNAc 4S-6ST and UST also disturbed the neuronal polarization of cultured hippocampal neurons, suggesting the importance of oversulfated chondroitin sulfate in this process. In the cultured hippocampal neurons, the oversulfated chondroitin sulfate was accumulated in the focal contacts in the cell bodies and axons. Chondroitinase ABC-treatment suppressed the tyrosine phosphorylation of FAK at the focal contacts, suggesting that the proteoglycans bearing oversulfated chondroitin sulfate strengthen the adhesion of axons and cell bodies to the substrate, leading to the stabilization of neuronal morphology. In contrast to the chondroitinase ABC-treatment, the axons extended steadily and showed almost no retraction when hippocampal neurons were treated with heparitinases that specifically degrade heparan sulfate. This suggests that heparan sulfate proteoglycans destabilize the neuronal morphology, inducing retraction of axons. In fact, it has been reported that heparan sulfate proteoglycans on growth cones are essential for the repulsive activities of Slit2 and ephrin-A3 (Hu, 2001; Irie et al., 2008). Thus, chondroitin sulfate and heparan sulfate proteoglycans expressed on hippocampal neurons play opposing roles during neuronal polarization.

# Roles of Glycosaminoglycans in Tangential Neuronal Migration

Cortical interneurons are born in the medial ganglionic eminence (MGE), caudal ganglionic eminence and preoptic area in the ventral telencephalon, and migrate tangentially toward the cortex (Evsyukova et al., 2013; Marin, 2013) (**Figure 4**). The newborn interneurons exiting the ganglionic eminence avoid entering the striatum, and migrate into the neocortex through the marginal zone, subplate, or lower intermediate/subventricular zones, suggesting that complex interplay of repulsive and attractive cues regulates the migration route of these neurons. Neuregulin-1, NT-4, and GDNF were shown to be chemoattractive factors for cortical interneurons, whereas Slit 1, Sema 3A, ephrin a3, and ephrin a5 act as chemorepulsive factors (Zhu et al., 1999; Polleux et al., 2002; Flames et al., 2004; Rudolph et al., 2010; Bespalov et al., 2011; Marin, 2013; Steinecke et al., 2014). As described above, biochemical studies revealed that these factors bind with chondroitin and/or heparan sulfate in a sulfation pattern-dependent manner. Thus, there is a possibility that chondroitin/heparan sulfate proteoglycans regulate the spatial distribution and/or activity of these factors. In fact, a recent report demonstrated that chondroitin sulfate plays an important role in the tangential migration of interneurons (Zimmer et al., 2010). Chondroitin sulfate proteoglycans are highly expressed in the striatal mantle zone, which is avoided by tangentially migrating interneurons (**Figure 5**). In vitro Boyden chamber cell migration and stripe assays demonstrated that chondroitin sulfate proteoglycans exert repulsive effects on cortical interneurons. These repulsive effects were suppressed by chondroitinase ABC-treatment, suggesting that chondroitin sulfate directly acts as a repellent for these neurons. Furthermore, in the chondroitinase ABC-treated brain slices, cortical interneurons actively invaded the striatum, although they avoided this region in the control slices. Sema3A is retained in the striatum by binding to the chondroitin sulfate chains, and repels migrating interneurons that express Sema3A receptor, neuropilin 1. Thus, it was shown that chondroitin sulfate proteoglycans exert not only direct, but also indirect repulsive effects on interneurons by anchoring repulsive factors in the striatum (**Figure 4**).

After the interneurons enter the neocortex, they avoid the cortical plate, where chondroitin sulfate proteoglycans are poorly expressed (**Figure 4**). Instead, they migrate through the chondroitin sulfate proteoglycan-rich marginal zone and subplate. They also prefer the subventricular zone and the lower intermediate zone, where the content of chondroitin sulfate proteoglycans is relatively low. Thus, it seems that chondroitin sulfate proteoglycans do not act as a repellent for interneurons in the neocortex. Tangential migration of interneurons in the neocortex is induced by the chemokine CXCL12 (SDF1), which is concentrated in the marginal zone, subplate, and lower intermediate/subventricular zones (Li et al., 2008; Lopez-Bendito et al., 2008). It has been reported that CXCL12 binds with both chondroitin sulfate and heparan sulfate (Mbemba et al., 2000). The marginal zone and subplate are highly enriched with phosphacan, neurocan and versican (Oohira et al., 1994; Maeda et al., 1995; Meyer-Puttlitz et al., 1996) (**Figure 5**), and thus CXCL12 may be anchored to the chondroitin sulfate chains of these proteoglycans. On the other hand, syndecan-1 is highly expressed in the ventricular/subventricular zone and in the lower intermediate zone (Wang et al., 2012), and may concentrate CXCL12 in these layers through heparan and/or chondroitin sulfate moieties. Thus, it seems that proteoglycans can be either attractive or repulsive substrates depending on the proteins bound to their glycosaminoglycan chains.

As described above, syndecan-3 functions as a GDNF receptor expressed on migrating interneuron (Bespalov et al., 2011). In this case, only the matrix-bound form of GDNF acts as a ligand of syndecan-3, and the soluble form is not active. It may be that GDNF bound to the chondroitin/heparan sulfate proteoglycans in the extracellular matrix activates the syndecan-3 signaling in the interneurons.

#### TABLE 1 | Human disorders caused by mutations of proteoglycan-related genes.

# Proteoglycans, Psychiatric Disorders, and Intellectual Disabilities

Since proteoglycans are major components of the connective tissue, mutations in the proteoglycan-related genes cause various skeletal and connective tissue disorders (Huegel et al., 2013; Mizumoto et al., 2013a). Recently, the involvement of these genes in intellectual and psychiatric disorders has also begun to be revealed (**Tables 1**, **2**). A hypofunctional mutation of XYLT1 encoding xylosyltransferase 1 causes an autosomal recessive short stature syndrome associated with intellectual disability (Schreml et al., 2014). Mutations of B3GALT6 encoding galactosyltransferase II cause a pleiotropic Ehlers-Danlos-syndrome-like connective tissue disorder, which is also associated with intellectual disability (Malfait et al., 2013). As described above, these two enzymes are involved in the biosynthesis of the linkage tetrasaccharides that are used commonly for the chain initiation of chondroitin and heparan sulfates, implying that these glycosaminoglycans are essential for the development of higher intellectual function of the brain. In fact, an earlier study suggested that EXT1, encoding a heparan sulfate co-polymerase, is associated with autism (Li et al., 2002). Loss-offunction mutations in CHSY1 encoding chondroitin synthase 1 cause Temtamy preaxial brachydactyly syndrome, which is characterized by delayed motor and mental development as well as bilateral, symmetric preaxial brachydactyly and hyperphalangism of digits, facial dysmorphism, and dental anomalies (Li et al., 2010). Furthermore, it was found that missense mutations of NDST1 cause intellectual disability, muscular hypotonia, and epilepsy, suggesting that normal modification of heparan sulfate is essential for the development of functional neuronal circuits (Reuter et al., 2014).

In addition to glycosaminoglycan biosynthetic enzymes, genes encoding proteoglycan core proteins are also associated with intellectual disability. Missense mutation in SPOCK1 encoding testican-1 causes intellectual disability with dyspraxia, dysarthria, partial agenesis of corpus callosum, and prenatal-onset microcephaly (Dhamija et al., 2014).

#### TABLE 2 | Proteoglycan-related genes proposed to be associated with mental disorders.

Simpson-Golabi-Behmel syndrome is an overgrowth/multiple congenital anomalies syndrome caused by mutations in glypican 3. This disease shows high clinical variability, and in some cases, intellectual disability is present (Tenorio et al., 2014). It will be important to examine whether these intellectual disabilities are caused by abnormal neuronal migration in the cortex.

It has recently been revealed that early developmental defects of neural network formation including abnormal neuronal migration and myelination can cause various psychiatric diseases such as schizophrenia (Stolp et al., 2012). Muhleisen et al. (2012) identified variation in the neurocan gene (rs1064395) as a common risk factor for bipolar disorder and schizophrenia. In schizophrenia patients, neurocan risk status was found to be associated with higher folding in the right lateral occipital cortex and left dorsolateral prefrontal cortex (Schultz et al., 2014). Neurocan may play important roles in the neuronal migration and/or formation of axonal fibers in the cerebral cortex, and the deficits in these processes may influence the folding of the occipital and prefrontal lobes, leading to an increased risk of schizophrenia.

It is well known that several members of the Neuregulin/ErbB signaling system are susceptibility genes of schizophrenia, bipolar disorders and depression (Mei and Nave, 2014). Neuregulins constitute a family of EGF-like signaling molecules that stimulate ErbB receptor family tyrosine kinases, the signaling of which regulates neuronal migration, myelination, neurotransmission and synaptic plasticity (Mei and Nave, 2014). Recent studies suggested that proteoglycans regulate Neuregulin/ErbB signaling, and thus are related to psychiatric disorders. Buxbaum et al. (2008) reported that PTPRZ1, which encodes both PTPζ and phosphacan, is associated with schizophrenia in a Caucasian population, although no association was found in the Japanese population (Ito et al., 2008). They demonstrated that PTPζ binds with ErbB4 through the scaffolding protein, MAGI, and inhibits the Neuregulin-1/ErbB4 signaling. Furthermore, Takahashi et al. (2011) found that the expression of PTPζ is increased in the brains of schizophrenia patients, and also demonstrated that transgenic mice overexpressing PTPζ showed reduced Neuregulin-1 signaling, and abnormal glutamatergic, GABAergic and dopaminergic activity as well as delayed oligodendrocyte development. In particular, it is remarkable that the number of parvalbumin-positive interneurons is decreased in the cortex of this transgenic mouse. Flames et al. (2004) demonstrated that loss of Neuregulin-1/ErbB4 signaling causes an alteration in the tangential migration of cortical interneurons and reduction in the number of GABAergic interneurons in the postnatal cortex. Therefore, PTPζ /phosphacan may negatively regulate Neuregulin-1/ErbB4 signaling, and inhibit the tangential migration of cortical interneurons.

Neuroglycan C was identified as a potential susceptibility gene for schizophrenia in a Southern Chinese population (So et al., 2010). As described above, neuroglycan C is involved in the radial neuronal migration in the neocortex (Zhang et al., 2013), and thus defects in this process may be involved in the etiology of schizophrenia. In addition, neuroglycan C has an EGF-like domain, acts as a direct ligand for ErbB3, and thus is classified as Neuregulin-6 (Kinugasa et al., 2004). It has been reported that ErbB3 is associated with schizophrenia in a Caucasian population (Li et al., 2009), and thus there is a possibility that neuroglycan C-ErbB3 signaling is involved in the pathophysiology of schizophrenia. ErbB3 plays important roles in oligodendrocyte differentiation and myelination (Mei and Nave, 2014), and neuroglycan C may regulate these processes. In this context, it is interesting to note that midkine-neuroglycan C signaling promotes process elongation of the oligodendrocyte precursor-like cell line, CG-4 (Ichihara-Tanaka et al., 2006).

In addition to proteoglycan core proteins, glycosaminoglycanmodifying enzymes have also been associated with psychiatric disorders. A genome-wide association study revealed that NDST3 is associated with schizophrenia and bipolar disorder, suggesting that the sulfation pattern of heparan sulfate plays an important role in the pathophysiology of these disorders (Lencz et al., 2013). Neuregulin-1 binds with heparan sulfate in a sulfation patterndependent manner, in which the N-sulfate group is the most important (Pankonin et al., 2005). Thus, the N-sulfated region in heparan sulfate may be important for normal Neuregulin-1/ErbB4 signaling.

The genomic region containing DSEL encoding dermatan sulfate epimerase 2 has been found to be associated with bipolar disorder (Goossens et al., 2003) and depressive disorder (Shi et al., 2011). In addition, it was reported that UST is associated with job-related exhaustion and response to antidepressant (Uher et al., 2010; Sulkava et al., 2013). Thus, chondroitin sulfate proteoglycans bearing oversulfated dermatan/chondroitin sulfate may play important roles in the etiology of mood disorders.

# Perspective

As described above, brain proteoglycans regulate the migration of both excitatory and inhibitory neurons by binding with various proteins. Besides neuronal migration, proteoglycans play important roles in the proliferation and differentiation of neural progenitor cells, axon pathfinding, myelination, axon regeneration, and maturation and plasticity of synapses (Maeda et al., 2011; Soleman et al., 2013; Silver and Silver, 2014; Theocharidis et al., 2014), the defects of which may be related to the pathogenesis of various brain disorders. More mechanistic studies are necessary to elucidate the relationship between proteoglycans and these diseases. In this context, it should be noted that many extracellular matrix proteins that interact with proteoglycans are overlooked in the field of developmental neuroscience. These include extracellular matrix proteases and their inhibitors such as MMP, ADAMTS, ADAM, and Timp family members. Proteoglycans turn over very rapidly in the developing brain, and their degradation would lead to drastic change in the distribution and activity of growth factors, chemokines, axon-guidance molecules, and so on. Thus, it is likely that degradation of proteoglycans would profoundly influence the behavior of neurons. Future study is necessary to shed light on this issue.

Finally, I would like to emphasize that the functions of proteoglycans are regulated in a context-dependent manner. It is often said that chondroitin sulfate proteoglycans are repulsive molecules. However, this over-simplified view has been challenged by the finding that chondroitin sulfate proteoglycans such as PTPζ and neuroglycan C promote the radial migration of cortical neurons. Furthermore, chondroitin sulfate proteoglycans may function as either a repulsive or an attractive substrate depending on the factors attached to the chondroitin sulfate chains. In addition, it should be noted that even though the proteoglycan core protein is the same, the structures of the attached glycosaminoglycan chains may be highly variable leading to the diversification of proteoglycan functions. I expect that careful experimental design and interpretation of the results would uncover the important functions of brain proteoglycans.

# References

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Maeda. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Neuronal and microglial regulators of cortical wiring: usual and novel guideposts

#### Paola Squarzoni\*, Morgane S. Thion and Sonia Garel\*

Centre National de la Recherche Scientifique UMR8197, Ecole Normale Supérieure, Institut de Biologie, Institut National de la Santé et de la Recherche Médicale U1024, Paris, France

Neocortex functioning relies on the formation of complex networks that begin to be assembled during embryogenesis by highly stereotyped processes of cell migration and axonal navigation. The guidance of cells and axons is driven by extracellular cues, released along by final targets or intermediate targets located along specific pathways. In particular, guidepost cells, originally described in the grasshopper, are considered discrete, specialized cell populations located at crucial decision points along axonal trajectories that regulate tract formation. These cells are usually early-born, transient and act at short-range or via cell-cell contact. The vast majority of guidepost cells initially identified were glial cells, which play a role in the formation of important axonal tracts in the forebrain, such as the corpus callosum, anterior, and post-optic commissures as well as optic chiasm. In the last decades, tangential migrating neurons have also been found to participate in the guidance of principal axonal tracts in the forebrain. This is the case for several examples such as guideposts for the lateral olfactory tract (LOT), corridor cells, which open an internal path for thalamo-cortical axons and Cajal-Retzius cells that have been involved in the formation of the entorhino-hippocampal connections. More recently, microglia, the resident macrophages of the brain, were specifically observed at the crossroads of important neuronal migratory routes and axonal tract pathways during forebrain development. We furthermore found that microglia participate to the shaping of prenatal forebrain circuits, thereby opening novel perspectives on forebrain development and wiring. Here we will review the last findings on already known guidepost cell populations and will discuss the role of microglia as a potentially new class of atypical guidepost cells.

Keywords: guidance molecules, glial cells, Cajal-Retzius cells, microglia, axon guidance

# Introduction

Functioning of the mammalian cerebral cortex relies on complex networks of axonal connections between neurons located in specific positions. The initial building of these exquisite circuits occurs during embryonic development and early post-natal days. During this "critical" period, neurons are first generated from spatially restricted proliferative niches and after or while reaching their final destination throughout active migration, extend oriented axons to form synaptic connections with their targets. Cellular migration is hence essential in the first part of brain wiring, because it allocates cells to specific positions and their subsequent settlement and differentiation, leading

#### Edited by:

Chiaki Ohtaka-Maruyama, Tokyo Metropolitan Institute of Medical Science, Japan

#### Reviewed by:

Umberto Di Porzio, Institute of Genetics and Biophysics "ABT", Italy Tatsumi Hirata, National Institute of Genetics, Japan

#### \*Correspondence:

Paola Squarzoni and Sonia Garel, Brain Development and Plasticity, Ecole Normale Supérieure, 46, rue d'Ulm, 75005 Paris, France<EMAIL_ADDRESS><EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neurogenesis, a section of the journal Frontiers in Neuroscience

Received: 13 May 2015 Accepted: 30 June 2015 Published: 17 July 2015

#### Citation:

Squarzoni P, Thion MS and Garel S (2015) Neuronal and microglial regulators of cortical wiring: usual and novel guideposts. Front. Neurosci. 9:248. doi: 10.3389/fnins.2015.00248 to the emergence of a functional system. In particular, cells can undertake radial or tangential migratory trajectories. During these processes, neurons can migrate locally or far away from their production sites as well as extend local axons or form long-range connections.

Because neurons are generated over a long-time period in the mammalian brain, neuronal migration and axonal navigation occur concomitantly during the constant process of brain development. How are these processes coordinated spatially and temporally to ensure the proper wiring of neural circuits? Over the last decades, this intriguing question has begun to receive answers in a developmental context in which cellular migration and axonal navigation take prominent places, namely the development of the embryonic mammalian forebrain (Borrell and Marin, 2006; Griveau et al., 2010; Villar-Cervino et al., 2013). In distinct regions of the embryonic mammalian forebrain, such as the dorsal cerebral cortex and the ventrally located subpallium, extensive events of radial and tangential migration reallocate neuronal populations and orient axonal navigation. For example, early-born neurons such as Cajal-Retzius cells spread out from different regions of origin to cover all the surface of the cortical primordium; inhibitory interneurons originate from the basal ganglia from which they tangentially migrate to populate the telencephalon; corticothalamic and thalamocortical axons traverse intermediate targets to reach their respective final targets. These events are fundamental to assemble cortical circuits and build the intricate circuitry essential for its functioning. Since defects in migratory processes during embryogenesis have been correlated with the onset of several neurologic and psychiatric diseases, it is crucial to decipher how they are regulated. Besides cells redistribution and morphogenesis, past and more recent studies showed that, throughout neuronal migration, an additional prominent event occurs during forebrain development that is the positioning of molecular cues, which instructs the trajectories of other migrating cells and growing axons. The cells that show these driving properties have different origins, but they share some common characteristics, for which they have been defined as guidepost cells. The purpose of this review is first to provide a definition of the usual concept of guidepost cells, giving an overview of already well-known examples. Moreover, we propose to extend the classical concept of guidepost cells, by speculating on recent findings concerning novel roles of microglia, the macrophages of the brain, in embryonic forebrain wiring.

# Toward a "Modern" Definition of Guidepost Cells

The concept of guidepost cells emerged from the studies on the developing limb bud of the grasshopper embryo (Borrell and Marin, 2006; Griveau et al., 2010; Kwon et al., 2011; Villar-Cervino et al., 2013). Bate and others described how pioneer projecting axons rely on some intermediate targets positioned along the future axonal path to follow a highly stereotyped pathway (Kwon et al., 2011). These intermediate targets consist of immature neuronal cells that show high affinity for the pioneer growth cones, and that are able, upon direct contact, to stabilize their filopodia and reorient the axonal growth cones on the pathway (Kwon et al., 2011). These important findings laid the foundation of the term "guidepost cells," as located discontinuously along the future axonal trajectory providing short-range cues thereby precisely controlling axonal navigation.

Since these seminal studies, several other cases of guidepost cells have been reported in different organisms and developmental systems (Borrell and Marin, 2006; Griveau et al., 2010; Kwon et al., 2011; Villar-Cervino et al., 2013). To date, the vast majority of the identified guidepost cells in vertebrates belongs to the class of glial cells, such as the radial glia of the optic chiasma (Misson et al., 1988; Guillery et al., 1995; Marcus et al., 1995; Marcus and Mason, 1995; Wang et al., 1995), glial bridges of anterior and postoptic commissures (Silver et al., 1982; Pires-Neto et al., 1998; Barresi et al., 2005; Lent et al., 2005), floor plate cells (Tessier-Lavigne et al., 1988; Bovolenta and Dodd, 1990, 1991; Placzek et al., 1990; Campbell and Peterson, 1993; Kennedy et al., 1994; Serafini et al., 1994, 1996), boundary cap cells (Golding and Cohen, 1997; Fraher et al., 2007), glial cells of the corpus callosum (Silver et al., 1982, 1993; Silver and Ogawa, 1983; Shu and Richards, 2001; Shu et al., 2003a,b).  Guidepost cells have been then defined as usually early born, discrete cell populations, with specialized functions that control and regulate axonal navigation, by being located at crucial decision points along the axonal trajectories. These cells can eventually extend an axon along the upcoming path of the tract and, in contrast to other long range-intermediate targets, guideposts act at short range or directly by cell-cell contact. They constitute decisive landmarks for guiding the axons along the correct pathways, which is a fundamental requirement for accurate circuitry assembly. The demonstration of their importance has been highlighted in different systems, by specific cell ablation experiments (Bentley and Caudy, 1983; Sretavan et al., 1995; Del Rio et al., 1997; Sato et al., 1998) and by the use of genetic mutants (Bovolenta and Dodd, 1990; Lopez-Bendito et al., 2006; Bielle et al., 2011), which resulted in aberrant pioneer axonal trajectories (Bielle et al., 2011), eventually with ectopic collateral branches formation (Bentley and Caudy, 1983; Bovolenta and Dodd, 1990) and in failure in axonal progression and in specific axonal innervation (Sretavan et al., 1995; Del Rio et al., 1997; Sato et al., 1998; Lopez-Bendito et al., 2006), respectively. The mechanism by which guidepost cells exert their function in guiding pioneer axonal tracts is throughout the secretion of guidance cues, which can act as attractive or repulsive signals.  The correct positioning of the same migrating guidepost cells at the intermediate targets along the path is itself instructed by guidance cues (Kawasaki et al., 2006; Nomura et al., 2006; Ito et al., 2008; Bielle et al., 2011). Since these cells act mainly at short range or by cell-cell contact, their proper localisation is fundamental for the subsequent axonal tract development. This has been clearly shown in various guidance molecule mutant models in which altered positioning of guidepost cells led to consequent specific axonal pathfinding defects (Kawasaki et al., 2006; Bielle et al., 2011).

# Tangential Migrating Guidepost Cells in the Pathfinding of the Lateral Olfactory Tract

The Lateral Olfactory Tract (LOT) is the main efferent axonal bundle that conveys the olfactory information from the bulb to several higher olfactory centers in the brain, including the anterior olfactory nucleus, the olfactory tubercle, the piriform and entorhinal cortices and the amygdala (Borrell and Marin, 2006; Griveau et al., 2010; Villar-Cervino et al., 2013) (**Figure 1**). Indeed, sensory olfactory neurons, residing in the nasal cavities project onto tufted and mitral cells of the olfactory bulb, which in turn extend their axons into the LOT to reach cortical and associated regions. LOT pioneer axons initiate their outgrowth around embryonic day (E) 11.5, followed by the other mitral cells that collectively form the main axonal bundle around E13. Starting from E14.5, LOT axons extend superficial collaterals toward the olfactory cortices and into the other target regions.

Long-range guiding activities are involved in shaping the pathfinding of the LOT. Diffusible repulsive guidance proteins, such as Slit1 and Slit2 derived from the septum, regulate the lateral pathfinding of the mitral cell axons, throughout their receptors, Robo1 and Robo2 (Pini, 1993; Nguyen Ba-Charvet et al., 1999; Nguyen-Ba-Charvet et al., 2002; Fouquet et al., 2007). Some proteins of the Semaphorin class are involved in the growth of olfactory bulb axons (Sema3B) and repulsion of LOT axons (Sema3F) (De Castro et al., 1999; De Castro, 2009). Besides these diffusible long-range signals, it was shown that a peculiar population of cells supplies short-range permissive guidance activity in the formation of the LOT (Sugisaki et al., 1996). These "lot" cells have been identified by the expression of the lot1 antibody (Sato et al., 1998), recently shown to recognize the beta isoform of the metabotropic glutamate receptor subtype-1 (mGluR1) (Hirata et al., 2012). Lot cells are the first reported example of migrating neuronal guidepost cells involved in axonal pathfinding and are amongst the first generated neurons in the brain, around E9.5 and E11.5. They have been proposed to have a pallial origin and migrate toward the pallial subpallial boundary (PSB) (Tomioka et al., 2000). Once arrived in the PSB, they change their orientation and extend a long process toward the amygdala region (Kawasaki et al., 2006; Hirata et al., 2012). They correspond to previously identified cells horizontally disposed in the developing PSB (Derer et al., 1977) and their positioning occurs way before the arrival of LOT axons (Sato et al., 1998), for which they constitute a growing substrate. Around E12.5,

the superficial growing of LOT axons displaces lot cells in the internal border of the path, where they are found subsequently in association with growing collateral axons (Hirata and Fujisawa, 1999). Although initially lot cells where considered as a distinct and unique cell population (Sato et al., 1998), these cells have been recently identified as a subset of Cajal-Retzius (CR) cells, a population of early born cortical neurons, since they share the expression of common molecular markers such as p 73 and Reelin (Dixit et al., 2014).

The role of CR-lot cells as guidepost for the developing LOT tract has been shown throughout toxic ablation experiments by the local use of a neuronal toxin, 6-hydoxydopamine, which provokes CR-lot cell death with consequent stall of mitral cell axons in strict proximity (Sato et al., 1998). This role is further highlighted by the analysis of mutant mice that affect this cell population such as Lhx2 (Saha et al., 2007) or Neurog1 and Neurog2 double mutants (Dixit et al., 2014). It has been proposed that lot cells form transient connections with LOT axons, as their final targets in the piriform cortex, amygdala and other higher olfactory centers are not yet mature (Sato et al., 1998; Hirata et al., 2012).

The importance of the proper positioning of CR-lot cells is thus highlighted by their guidepost function to orient LOT axons along their pathway. Still, how these cells act on axons and whether they are required for the progression, channeling or guidance of all axons remains largely to be characterized. By contrast, several guidance cues have been shown to play a role in the positioning of CR-lot cells in the ventral PSB. Netrin1 has been shown to act as an attractant cue for migrating CR-lot cells and participates, in part, to their ventral positioning (Kawasaki et al., 2006). However, in knockout mutant animals for Netrin1 or its receptor DCC, only the location of the most ventral CRlot cells resulted affected, associated with specific pathfinding defects on the ventral most LOT axons (Kawasaki et al., 2006). In double Slit1; Slit2 mutants the LOT axonal tract is severely disrupted, with only few axons present in their correct positions. In this context, the proper positioning of CR-lot cells appears to be not drastically affected, thereby revealing that both longrange and local signals cooperate in LOT axonal pathfinding (Fouquet et al., 2007). Another important regulator of the ventral tangential migration of CR-lot cells is the molecule Sema3F that, by the interaction with its specific receptor neuropilin-2 (Nrp-2), confines CR-lot cells on the telencephalic surface (Ito et al., 2008). Sema3F, expressed in the subpallium and cortical plate, acts as a repellent signal, which prevents CR-lot cells to penetrate into deep brain regions, where some are ectopically found in case of Sema3F or Nrp-2 invalidation (Ito et al., 2008). So far, there are not yet reported defects of LOT projections in Nrp-2 mutants (Chen et al., 2000), raising the possibility that these guidepost cells may act locally. Furthermore, since many of these guidance cues can directly act on the axons, additional eventual effects of these genetic invalidations on the pathfinding of LOT axons deserve further analyses.

# Cajal-Retzius Cells: Guideposts in the Formation of Entorhino-Hippocampal Projections

Besides their emerging role in LOT axonal guidance, Cajal-Retzius cells, together with GABAergic interneurons, have been involved in the development of entorhino-hippocampal projections (Borrell and Marin, 2006; Griveau et al., 2010; Villar-Cervino et al., 2013). The major afferent excitatory projections in the hippocampus derive from pyramidal neurons in layers II and III of the entorhinal cortex. In particular, layer II pyramidal neurons form axonal connections with the dendrites of the granule cells of the outer molecular layer (OML) of the dentate gyrus (DG), whereas layer III neurons connect mainly with pyramidal cells in the stratum lacunosum-moleculare (SLM) in the cornu ammonis 1 and 3 (CA1 and CA3) (Borrell and Marin, 2006; Griveau et al., 2010; Villar-Cervino et al., 2013). Notably, during brain formation, the entorhinal axons already reach their final positions in the hippocampal regions, before the definitive development of their targets. Indeed, in mouse brain, entorhinal axons arrive in the hippocampus around E15, then they form arborisations in the SLM around E17 and are detected into the OML starting from the first postnatal day (Super and Soriano, 1994; Super et al., 1998; Deng and Elberger, 2001; Deng et al., 2006) (**Figure 2**). Therefore, even if hippocampal pyramidal neurons and granule cells are generated between E14 and E16, it is only around the second postnatal day that their apical dendrites start to be seen in the SLM, arising as final targets for entorhinal axons (Caviness, 1973; Soriano et al., 1986, 1989; Bayer and Altman, 1987; Super et al., 1998). This process of precise axonal addressing is regulated by Cajal-Retzius cells, which, as in LOT formation, have been reported to regulate axonal outgrowth. Cajal-Retzius (CR) cells are early born neurons, which are produced at E9-11 by focal pallial sources, including cortical hem, septum, PSB, and thalamic eminence (Grove et al., 1998; Meyer et al., 1999, 2002; Meyer and Wahle, 1999; Hevner et al., 2003; Takiguchi-Hayashi et al., 2004; Bielle et al., 2005; Cabrera-Socorro et al., 2007; Tissir et al., 2009; Ceci et al., 2010; Meyer, 2010; Gu et al., 2011; Martinez-Cerdeno and Noctor, 2014). CR cells migrate tangentially from their sources in the marginal zone of the cerebral cortex and rapidly cover the entire sheet. Their marginal localization and migration is regulated by CXCL12 produced by the meninges, which acts through CXCR4 and CXCR7 receptors (Borello and Pierani, 2010; Trousse et al., 2014). The marginal maintenance of CR cells also requires radial glia integrity, as revealed by

analysis of β1 integrin conditional knockout (Kwon et al., 2011). In parallel, interactions between CR cells or with surrounding structures have been shown to control their random dispersion and distribution by Eph/ephrin-dependent contact repulsion or PlexinD1 signaling (Villar-Cervino et al., 2013; Bribian et al., 2014). Such migratory behaviors enable CR cells of different sources to preferentially cover cortical regions (Griveau et al., 2010; Gu et al., 2011). Functionally, CR cells have been shown to regulate cortical layering, neuronal and radial glia morphology, and cortical regionalisation, via the production of the secreted glycoprotein Reelin or additional membrane-bound or secreted factors (Borello and Pierani, 2010; Griveau et al., 2010; Gil-Sanz et al., 2013; Trousse et al., 2014). In the hippocampus, as the structure folds during development, CR cells localize in the future SLM and OML (Takiguchi-Hayashi et al., 2004; Bielle et al., 2005; Yoshida et al., 2006). Using electron microscopy, it has been shown that during embryogenesis pioneer entorhinal axons form transient synaptic contacts with Cajal-Retzius cells in SLM and OML, the future regions that will host pyramidal and granule cells apical dendrites (Super et al., 1998). Moreover, in organotypic culture experiments, this interaction has been shown to be fundamental for the growth of entorhinal axons in the hippocampus (Frotscher and Heimrich, 1993; Li et al., 1993; Frotscher et al., 1995; Del Rio et al., 1997). Indeed, eliminating CR cells from cultured slices with the local toxin 6-hydoxydopamine avoided entorhinal axonal growing in the hippocampus (Del Rio et al., 1997). These experiments therefore constituted a strong evidence of the role as placeholders for CR cells in the formation of entorhino-hippocampal connections (Forster et al., 1998). About the molecular cues that could be involved in entorhinal axon guidance by CR cells, the most obvious candidate could be the glycoprotein Reelin, of which CR cells constitute the main source (D'arcangelo et al., 1995, 1997; Hirotsune et al., 1995; Ogawa et al., 1995; Tissir and Goffinet, 2003). It has been previously shown that Reelin controls cortical layering, organization and the orientation of radially migrating neurons (Borello and Pierani, 2010; Frotscher, 2010; Griveau et al., 2010; Martinez-Cerdeno and Noctor, 2014). Nevertheless, antibody-mediated blocking of Reelin in ex vivo co-cultures of hippocampal slices and entorhinal tissue, does not lead to dramatic defects in entorhinal axonal pathfinding. However, fewer entorhinal fibers reach the hippocampal layers, developing shorter axonal branches. These findings have been confirmed in vivo in reeler mice, a natural Reelin mutant, which presents severe defects in cortical lamination. Similarly to co-cultures experiments, the absence of Reelin had no dramatic effects about entorhinal axonal ingrowth or targeting, but entorhinohippocampal axonal terminations appear thinner than in control animals. Moreover, these defects are transient, since then in reeler adult mice a normal branching density is observable (Frotscher and Heimrich, 1993; Li et al., 1993; Frotscher et al., 1995; Del Rio et al., 1997; Borrell et al., 1999; Deller et al., 1999). Altogether, these results confirm the important role of CR as guidepost cells in entorhino-hippocampal innervation and reveal reelin as an important factor for branching, collateral formation and synaptogenesis of entorhinal axons. However, they leave still an open question about additional molecular cues involved in the pathfinding of entorhinal axons in the hippocampus.

# En Route to the Cortex: Guidepost Cells Open a Path for Thalamocortical Connections

Mammalian neocortex forms connections with the rest of the brain via the internal capsule, which includes bundles of corticofugal efferent axons and reciprocal afferent thalamocortical projections, which convey sensory and motor information to the neocortex. In the context of axonal pathfinding, the development of the internal capsule has been extensively studied (Molnar et al., 2012; Garel and Lopez-Bendito, 2014). Indeed, this system has a major physiological relevance, but also allows a variety of experimental approaches, due to its important size and extension in the developing brain.

During the years, many findings had contributed to elucidate the routes and the molecular mechanisms that shape thalamocortical and corticofugal connection paths. In mouse development, these important axonal systems start to form during early/mid gestation. Thalamocortical axons (TCAs) originate from neurons located in the thalamus, grouped in distinct nuclei, showing a topographic organization that corresponds to the spatial innervation of different cortical areas (Molnar et al., 2012; Garel and Lopez-Bendito, 2014). From E12 to E15, TCAs extend ventrally, crossing the prethalamus, and traverse the diencephalic/telencephalic boundary, entering the subpallium at the level of the internal capsule. At E14, early TCAs reach the PSB, where they encounter the reciprocal pioneer corticothalamic axons (CTAs). Subsequently, from E14.5 to E18.5, TCAs form transient connections with subplate cells residing in their respective target cortical areas. After this waiting period, TCAs send collaterals into the cortical plate and finally establish thalamocortical connections (**Figure 3**). Meanwhile, corticofugal axons grow along the same path of TCAs and split in CTAs and in corticosubcerebral axons that proceed toward other subcortical regions. This reciprocal wiring has been shown to be tightly controlled, in part by guidepost cells, transient axonal populations and several structures that have been shown to act as milestones along the path.

Chronologically, pioneer cortical subplate neurons have been firstly proposed as guidepost cells in regulating the entering and progression of TCAs into the cortical plate (Garel and Lopez-Bendito, 2014; Hoerder-Suabedissen and Molnar, 2015). These observations have been strongly supported by several experimental evidences. To date, in the visual cortex, subplate neurons ablation avoids the entering of the corresponding thalamic geniculocortical axons (Ghosh et al., 1990; Ghosh and Shatz, 1993). In mutant mice, such as reeler, p35−/<sup>−</sup> and cdk5−/<sup>−</sup> that present subplate cells in the marginal zone, due to severe defects in preplate splitting, TCAs form abnormal projections to connect with the ectopic subplate in the marginal zone. Since the discovery of subplate cells, other groups of cells have been found to exert a role of guidepost for TCAs. In mouse, early born cells, named perireticular cells, have been identified in the

future path of the internal capsule, which at E12.5 send a cellular process to the thalamus. The hypothesized role of these cells is to provide a cellular scaffold for the future TCAs and CTAs, which is consistent with several experimental evidences (Mitrofanis, 1992, 1994; Mitrofanis and Baker, 1993; Mitrofanis and Guillery, 1993; Adams and Baker, 1995; Metin and Godement, 1996; Molnar et al., 1998b; Braisted et al., 1999; Molnar and Cordery, 1999). In effect, different defects in TCA pathfinding have been reported in mutant mice presenting absence, reduction or displacement of perireticular cells (Tuttle et al., 1999; Bishop et al., 2000, 2003; Lopez-Bendito et al., 2002; Lakhina et al., 2007). Unfortunately, the current absence of specific molecular markers and the wide distribution of these cells experimentally limit the investigation about their origin and function.

More recently, another population of guidepost cells has been observed in the subpallium, which comprises the lateral and medial ganglionic eminences (LGE and MGE). These cells are GABAergic LGE derived-neurons that tangentially migrate in the MGE, forming a permissive corridor, in an otherwise not permissive territory, for the growth of TCAs along an internal path toward the cortex (Lopez-Bendito et al., 2006). Because of their function, they have been named "corridor" cells; they are located in the MGE, in which they migrate from E11.5 to E14, but express LGE molecular markers, such as Islet1, Ebf1 and Meis2. By gain-of-function experiments in cultured organotypic embryonic brain slices and by the use of full or conditional mutant mice of ErbB4 and Neuregulin1 respectively, it has been revealed that corridor cells, via the expression of Neuregulin1, provide a permissive corridor for TCAs, which express the corresponding ErbB4 receptor (Lopez-Bendito et al., 2006). How are corridor cells positioned? A ventral repulsive activity from the subpallium, mediated by Slit2 and Robo1 and Robo2 respective receptors, has been shown to limit, in vitro, ex vivo, and in vivo, the ventral tangential migration of corridor cells, playing a role in the formation of corridor shape. Indeed, Slit2 inactivation leads to abnormal ventral migration of corridor cells, with aberrant corridor shaping and consequent defects on TCAs pathfinding (Bielle et al., 2011). These findings constitute a starting point for further investigations on the origin, specification and fate of corridor cells.

In addition to guidepost cells, several important structures and molecules present in the subpallium or prethalamus have been shown to play critical roles in the guidance of both TCAs and reciprocal CTAs (Metin and Godement, 1996; Braisted et al., 1999; Garel et al., 1999; Sussel et al., 1999; Marin et al., 2002; Marin and Rubenstein, 2002; Yun et al., 2003). For instance, knockout mutation analyses, have highlighted the importance of classical guidance cues and their receptors, such as Slit1, Slit2, Robo1, Robo2, Netrin1, and Sema6A, for the local regulation of TCAs guidance (Braisted et al., 2000, 2009; Leighton et al., 2001; Bagri et al., 2002; Bonnin et al., 2007; Lopez-Bendito et al., 2007; Powell et al., 2008; Little et al., 2009). Other important regulators are the members of the protocadherin family, which have been shown to be important for both TCAs and CTAs progression into the subpallium (Wang et al., 2002, 2006; Tissir et al., 2005; Uemura et al., 2007; Zhou et al., 2008, 2009; Qu et al., 2014). For instance, inactivation of OL-protocadherin was shown to impair the subpallial crossing of TCA associated with defects in striatal axonal outgrowth (Uemura et al., 2007). In addition, mutant mice with constitutive or specific inactivation of Celsr3 in the prethalamus and subpallium present similar impairments, which consist in the stall of TCAs in the ventral subpallium, across the diencephalic/telencephalic boundary, and in CTAs arrest in the proximal part of the LGE, suggesting a possible cooperation of these two factors in the process (Tissir et al., 2005; Zhou et al., 2008, 2009; Qu et al., 2014). Strikingly, these phenotypes are almost phenocopied by deletion of Frizzled3, which is associated with Celsr signaling pathway (Wang et al., 2002, 2006). Last, but not least, the expression of the transmembrane protein Linx is required on subplate cells, subpallium and prethalamaus guideposts, for the progression of TCAs and CTAs revealing a role for this molecule in axon/axon interactions and potentially guideposts/axons interactions (Mandai et al., 2014). More generally, it will be essential to precise which of the aforementioned guidance cues regulates the positioning and/or function of guidepost cells located along the internal capsule path.

In addition to delineating an internal trajectory for TCAs, guidepost neurons have been shown to play additional roles in thalamo-cortical wiring. First, there is now solid evidence that TCAs and CTAs interact to form reciprocal connections, as proposed by the handshake hypothesis (Blakemore and Molnar, 1990; Molnar and Blakemore, 1991, 1995; Chen et al., 2012; Molnar et al., 2012; Deck et al., 2013; Garel and Lopez-Bendito, 2014). As such, guideposts that shape TCAs path have an indirect impact on the guidance of reciprocal CTAs. Second, while TCAs originating from principal thalamic nuclei all grow internally in the corridor, they adopt distinct rostrocaudal positions in the capsule, depending on their nucleus of origin and their cortical target (Molnar et al., 1998a, 2012; Garel and Lopez-Bendito, 2014).  Indeed, guidance factors such as Slit1 and Netrin1 (and their combinatorial activity), Sema3A, ephrinAs, as well as L1, CHL1 participate to the topographic ordering of TCAs deriving from different thalamic nuclei, with a dramatic impact on their final cortical addressing (Bonnin et al., 2007; Wright et al., 2007; Powell et al., 2008; Bielle et al., 2011; Demyanenko et al., 2011a,b; Lokmane et al., 2013). Remarkably, positional information has been shown to be present already in the corridor, as TCAs enter the subpallium (Bielle et al., 2011). Accordingly, the aforementioned guidance factors are present in the corridor, especially Slit1, supporting the idea that they act as TCAs grow internally (Bielle et al., 2011). Importantly, altering the ordering of TCAs in the subpallium by genetic manipulation has been shown to impair the fine topography of TCAs in the somatosensory cortex (Lokmane et al., 2013; Lokmane and Garel, 2014). These experiments reveal that in addition to cortical signals, intermediate ordering of axons, in part by corridor cells, is important for fine-grained topography of TCAs. Together, such recent studies highlight additional roles of corridor internal guideposts in reciprocal and topographical wiring.

# Microglia Cells: Novel Unusual Guidepost Cells?

Microglia are the resident macrophages of the brain, which control brain homeostasis in physiologic conditions and constitute the first line of defense in case of diseases and against pathological threats. Initially described by Del Rio-Hortega (1932), the physiological functions and the origin of these cells have been remained controversial for a long time. Until recently, most studies focused on the roles of microglia in brain damage and diseases and in their participation to neuro-inflammatory processes via the release of neurotrophic and pro-inflammatory factors as well as the ability to perform phagocytosis. Over last decade, several landmark studies have revealed that, using conserved cellular mechanisms, microglia contribute to normal brain functions. Indeed, microglia have been shown to modulate synaptic transmission, to regulate synaptic formation and elimination, and to shape postnatal and embryonic brain circuits as reviewed in Paolicelli and Gross (2011), Schafer et al. (2013), Bilimoria and Stevens (2014), Katsumoto et al. (2014), Paolicelli et al. (2014), Salter and Beggs (2014) and Casano and Peri (2015). Below, we will focus on specific features of microglia during early brain wiring that bear similarities with those of guideposts cells, such as their capacity to act at short range, their early origin and focal positioning as well as the production of several molecular factors by which they can interact with and condition their surrounding neural environment.

# Microglia Survey and Interact with Their Local Environment

In the last decade, technological advancements such as twophoton laser scanning microscopy, allowed the observation of microglia behavior in vivo, in normal conditions. Ramified microglia, initially thought to be in a resting state in opposition to the activated, amoeboid morphology observed following brain injury, were found to be extremely active in surveying their environment. Indeed, very frequent extensions and retractions of microglia ramifications were observed in contact with neighboring neuronal cells, astrocytes and blood vessels; furthermore their extensions were increased by changes in both neuronal activity, blood vessel lesions and ATP variation levels in vivo (Davalos et al., 2005; Nimmerjahn et al., 2005). Processes of "resting microglia" were found to interact with synapses in somatosensory and visual neocortex, forming direct appositions with different synaptic elements. In particular, morphological changes of microglia processes, with the appearance of phagocytic structures and modifications of synaptic apposition frequencies were shown to be modulated by variation in visual experience (Wake et al., 2009; Tremblay et al., 2010). Following similar lines, microglia were found to modulate neuronal activity (Li et al., 2012; Pascual et al., 2012). For instance, in zebrafish larvae optic tectum, microglia were shown to contact highly activated neurons for longer time, correlating with a subsequent decreased neuronal activity (Li et al., 2012). Conversely, microglia activation in vitro by LPS stimulation was reported to indirectly increase the frequency of spontaneous synaptic AMPAergic post-synaptic currents in hippocampal neurons (Pascual et al., 2012). Such findings have fundamentally changed our conception of microglia by revealing that these cells exert the capacity to act at short-range on their surrounding neural environment. Since then, besides their immune-defensives functions, these cells have started to be considered as active modulators during healthy brain development and maturation as well as actors of pathologic brain wiring and functioning.

# Early Origin of Microglia

Originally thought to arise form peripheral bone marrow derived-macrophages that invade the brain after birth, microglia have been show, throughout series of fate-mapping experiments, to originate from yolk sac myeloid progenitors and to be dependent on Pu.1, Irf8 (Kierdorf et al., 2013) and colonystimulating factor 1 receptor (CSF1R) (Ginhoux et al., 2010, 2013; Erblich et al., 2011; Schulz et al., 2012; Gomez Perdiguero et al., 2013; Kierdorf et al., 2013; Hoeffel et al., 2015). In mice, yolk sac derived-microglia precursors migrate into the neural folds during embryogenesis and, by in situ proliferation, generate microglia that populate the adult brain. Under normal conditions, microglia comprise resident cells since the infiltration of peripheral monocytes or macrophages into the CNS is limited by the blood-brain barrier (Mildner et al., 2007; Ginhoux et al., 2010; Schulz et al., 2012; Gomez Perdiguero et al., 2013). Thus, microglia enter the brain from early prenatal stages and form an autonomous, self-sustained population. Remarkably, colonization of embryonic brain tissues by microglia appears to be a highly conserved process across vertebrate species (Perry et al., 1985; Ashwell, 1991; Cuadros and Navascues, 2001; Herbomel et al., 2001; Verney et al., 2010; Schlegelmilch et al., 2011; Swinnen et al., 2013), suggesting that embryological "seeding" of the microglial population may be also conserved.

How is the number or density of microglia regulated? Different embryonic or postnatal methods have been reported for the ablation of microglia in vivo or in cultured brain slices (Duffield et al., 2005; Heppner et al., 2005; Varvel et al., 2012; Ueno et al., 2013; Elmore et al., 2014; Squarzoni et al., 2014). Among those methods, pharmacologic depletion models acting on CSF1R signaling, revealed that after birth, a complete microglia repopulation occurs in a 1-week time window (Elmore et al., 2014; Squarzoni et al., 2014). These results show that there is a homeostatic control over the microglial population and raise the questions of the underlying mechanisms. While the origin of these repopulating cells is still debated, it has been shown for the adult repopulation that a local brain pool of nestin-positive cells differentiates into microglia thereby restoring their usual number (Elmore et al., 2014). Collectively, these essential findings match some forward-looking theories formulated by del Rio Hortega, which postulated that microglia enter the brain during embryogenesis (Del Rio-Hortega, 1932); at the same time, they highlight how the constant presence of microglia within the brain is tightly regulated.

# Microglia in Defining the Number of Neurons: Neurogenesis and Survival

Microglia have been recently shown to take part to several important events which contribute to shaping of neural circuits, including neurogenesis, neuronal survival, synaptic remodeling and maturation. The role in neurogenesis and survival has been examined in both the adult niche and the developing brain. For instance, in adult murine hippocampus, unchallenged microglia regulate by phagocytosis the number of immature neurons maintained in the subventricular zone, one of the few sites of postnatal neurogenesis (Sierra et al., 2010, 2013). In macaque and rat neocortex, alteration of microglia activity by maternal immune activation through LPS, Doxycycline treatment or microglia elimination by Liposomal clodronate exposition, significantly affects the number of neuronal precursors in the embryonic and postnatal brain (Cunningham et al., 2013).

In parallel, microglia have been also reported to regulate neuronal number by active induction of apoptosis, or oppositely to contribute to neuronal survival, in different regions of the brain. For instance, early postnatal apoptosis in the cerebellar Purkinje cell (PC) population was shown to be induced by superoxide ions generated from microglial respiratory bursts (Marin-Teva et al., 2004). These results provided support to previous studies showing that the depletion of microglia in brain culture slices in vitro resulted in increased PC survival (Van Rooijen et al., 1997). Likewise, in perinatal mouse hippocampus, microglia was found to enhance hippocampal neuronal apoptosis by the CD11b/DAP12 integrin signalingdependent production of reactive oxygen species (Wakselman et al., 2008). Conversely, microglia have been shown to actively sustain postnatal cell survival of layer V cortical neurons in mouse by the production of the trophic factor IGF1 (Ueno et al., 2013). Indeed, postnatal microglia inactivation by minocycline, microglia temporal elimination in CD11b-DTR transgenic models, as well as the use of IGF1R inhibitors and igf1 siRNA, resulted in increased cell death of layer V cortical neurons (Ueno et al., 2013). Altogether, these findings show that microglia regulate the number of neurons produced and maintained in the brain, through a balanced activity on progenitors, immature neurons and maturing neurons.

# Microglia Shape the Postnatal Brain: Synapse Formation and Synapse Pruning

Besides their roles on neurogenesis and neuronal cell homeostasis, microglia have been found to contribute to synaptogenesis, synaptic remodeling and brain maturation. Similarly to IGF1 for layer V cortical neurons, the production of the neurotrophin BDNF by microglia has been shown to promote synapse formation via signaling to its cognate receptor TrkB. Remarkably, specific microglia or microglia-BDNF depletion, using CX3CR1CreER mice, both lead to deficits in multiple learning tasks and learning-induced synaptic remodeling (Parkhurst et al., 2013). These major findings highlight the ability of microglial cells to impact on the building and homeostasis of neural circuits throughout the very local active production of secreted factors.

In addition, microglia were shown to play an active role in postnatal synaptic pruning, contributing to the shaping and maturation of the brain, by their close spatial and temporal contact with synapses (Paolicelli et al., 2011; Schafer et al., 2012, 2013; Kettenmann et al., 2013). In particular, this has been directly observed in the retinogeniculate system, where surrounding microglia participate to the activitydependent synaptic remodeling, eliminating the weaker presynaptic connections through a C3/CR3 complementdependent mechanism (Schafer et al., 2012, 2013). Similarly, in the hippocampus microglia were found to contribute to synaptic refinement. Specifically, the CX3CR1/fractalkine signaling pathway plays a central role in microglia/synapses communication, since Cx3cr1−/<sup>−</sup> mice show temporal reduction of hippocampal microglia number, leading to a deficit in synaptic pruning. Consistently, with early defects in synaptic communication, these mice were shown to exhibit reduced functional brain connectivity, together with social interaction and behavioral deficits (Paolicelli et al., 2011; Zhan et al., 2014). In the somatosensory neocortex, reduced density of microglia cells of Cx3cr1−/<sup>−</sup> mice due to a delay in recruitment of these cells, has been shown to impact on the maturation of thalamocortical synapses (Hoshiko et al., 2012). Thus, the density of these cells, their proper functioning, as well as their capacity to specifically perform local phagocytosis or production of secreted factors, constitutes an important factor for sculpting postnatal brain circuits.

# Microglia in the Embryonic and Perinatal Brain: the Importance of Spatial and Temporal Positioning

What about a role of microglia during embryogenesis? In contrast to their later homogeneous distribution in the adult brain, embryonic, and perinatal microglia show an uneven distribution in different species (Ashwell, 1991; Verney et al., 2010; Arnoux et al., 2013; Cunningham et al., 2013; Swinnen et al., 2013; Squarzoni et al., 2014). In particular, in the mouse, round or more ramified microglia have been observed in different focal hotspots, which are not particularly related to apoptosis, whereas some zones, such as the cortical plate is largely devoid of microglial cells (Ashwell, 1991; Verney et al., 2010; Cunningham et al., 2013; Swinnen et al., 2013; Squarzoni et al., 2014). More in depth analyses revealed that microglia accumulations correspond to important decision landmarks in axonal paths or cellular migratory routes. In particular, discrete groups of microglia associate with the corpus callosum, the external capsule or establish a contact with incoming dopaminergic axons in the ventral telencephalon. Specific associations with progenitor zones have also been observed in mice and other mammals, potentially regulated by chemokine production by these progenitors (Cunningham et al., 2013; Arno et al., 2014).

Pharmacologic or genetic ablations of microglia have been used to probe the roles of these cells during embryonic brain wiring (**Figure 4**). Together with maternal immune activation (MIA) and genetic microglial impairment (Cx3cr1−/−), these studies showed that microglia regulate the outgrowth of dopaminergic axons, thereby revealing the importance of the precise spatial-temporal microglia localisation (Squarzoni et al., 2014). In addition, microglia contribute to the development of the Corpus Callosum (CC), the largest commissural structure between the cerebral hemispheres (Pont-Lezica et al., 2014). Indeed, genetic functional impairment of microglia (Dap12−/−) or developmental functional alteration by MIA, down-regulate the expression of genes related to neuritogenesis in microglia, with a consequent impairment on the CC fasciculation in these mouse models. A similar CC fasciculation phenotype has been equally observed in the genetic model of microglia ablation, Pu·1 <sup>−</sup>/<sup>−</sup> (Pont-Lezica et al., 2014). Together these studies suggest that the spatial and temporal positioning of embryonic microglia modulates the development of specific and important axonal tracts. The underlying cellular and molecular mechanisms still remain to be deciphered.

In addition, microglia, which show a timely invasion of the cortical plate (CP) (Cunningham et al., 2013; Swinnen et al., 2013; Squarzoni et al., 2014) were found to regulate the assembly of cortical circuits. Cortical circuits are formed by an intricate network of a majority of excitatory neurons and a minority of functionally important inhibitory interneurons (Marin and Rubenstein, 2003; Sur and Rubenstein, 2005; Batista-Brito and Fishell, 2009; Cossart, 2011; Fishell and Rudy, 2011; Rico and Marin, 2011; Rubenstein, 2011; Marin and Muller, 2014). Indeed, various classes of interneurons shape the network output and interneuron dysfunction as well as defects in the excitation/inhibition balance have been associated with several neurodevelopmental disorders such as Autism Spectrum Disorders (ASD) or Schizophrenia. As aforementioned, microglia regulate the number of neuronal precursors in the subventricular zones of the neocortex (Cunningham et al., 2013) and are firstly excluded from the CP, which they invade after E16.5, remaining initially confined to the deeper layers (Swinnen et al., 2013; Squarzoni et al., 2014). Absence, immune activation or genetic impairment of microglia were found to impact on the laminar distribution of a specific population of interneurons that express the transcription factor Lhx6. Indeed, in absence of microglia (pharmacological

depletion; Pu·1 <sup>−</sup>/−), or in case of pharmacological (MIA) or genetic functional alteration (Dap12−/−; Cx3cr1−/−), a premature entry of lhx6-expressing interneurons in the CP was observed, followed by an altered laminar distribution, with long lasting postnatal effects on a subset of lhx6 expressing interneurons, the fast-spiking parvalbumin-positive interneurons (Squarzoni et al., 2014). These specific interneurons have been shown to play a major role in cortical networks as well as to be impaired in ASD and Schizophrenia (Penagarikano et al., 2011; Marin, 2012; Meechan et al., 2012). While these results reveal a surprising role of microglia in cortical circuits assembly, as well as a potential involvement in the etiology of neuropsychiatric diseases, they raise the question of the underlying mechanisms. Besides the requirement of Dap12 and Cx3cr1 signaling, the processes involved deserve further investigation.

These results reveal that microglia modulate brain wiring at various developmental steps, starting from embryonic, post-natal and adult stages. Moreover, they underlie the importance of spatial and temporal positioning of these cells to accomplish their roles as modulators of dopaminergic axonal outgrowth, CC development and neocortical interneuron laminar distribution, which are major events in forebrain wiring.

# Conclusions and Perspectives

While the concept of guidepost cell has substantially changed since its first description, including a potential motility as well as a diverse cellular identity (neuronal or glial), there are still some conserved properties: they are usually early born, immature, located at a crucial point along a pathway and able to act at short-range or by direct cell-cell contact on its target. Along these lines, recent studies have revealed that microglia cells may be to some extent, envisaged as novel guideposts during embryonic forebrain wiring. By their transient specific localization during embryogenesis they may act on restricted neuronal subgroups and modulate forebrain wiring. If a comprehensive knowledge of all microglia functions is still fragmentary, the tremendous potential of these cells in shaping and remodeling circuits, during normal and pathological conditions, opens a novel framework for our understanding of brain wiring.

# References

# Acknowledgments

We are grateful to members of the Garel lab for stimulating inputs, discussions and critical comments on the manuscript. The lab is supported by Investissements d'Avenir implemented by ANR-10-LABX-54 MEMO LIFE, ANR-11-IDEX-0001-02 PSL<sup>∗</sup> Research University, the ANR-12-BVS4-0010-01 Corridor and ERC Consolidator NImO. SG is an EMBO YIP awardee and part of the Ecole des Neurosciences de Paris Ile-de-France network.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Squarzoni, Thion and Garel. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Cellular dynamics of neuronal migration in the hippocampus

Kanehiro Hayashi, Ken-ichiro Kubo, Ayako Kitazawa and Kazunori Nakajima\*

*Department of Anatomy, Keio University School of Medicine, Tokyo, Japan*

A fine structure of the hippocampus is required for proper functions, and disruption of this formation by neuronal migration defects during development may play a role in some psychiatric illnesses. During hippocampal development in rodents, pyramidal neurons in the Ammon's horn are mostly generated in the ventricular zone (VZ), spent as multipolar cells just above the VZ, and then migrate radially toward the pial surface, ultimately settling into the hippocampal plate. Although this process is similar to that of neocortical projection neurons, these are not identical. In addition to numerous histological studies, the development of novel techniques gives a clear picture of the cellular dynamics of hippocampal neurons, as well as neocortical neurons. In this article, we provide an overview of the cellular mechanisms of rodent hippocampal neuronal migration including those of dentate granule cells, especially focusing on the differences of migration modes between hippocampal neurons and neocortical neurons. The unique migration mode of hippocampal pyramidal neurons might enable clonally related cells in the Ammon's horn to distribute in a horizontal fashion.

#### Edited by:

*Perry Bartlett, University of Queensland, Australia*

#### Reviewed by:

*Tatsunori Seki, Tokyo Medical University, Japan Toshio Ohshima, Waseda University, Japan*

#### \*Correspondence:

*Kazunori Nakajima, Department of Anatomy, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Neurogenesis, a section of the journal Frontiers in Neuroscience*

Received: *31 January 2015* Paper pending published: *01 March 2015* Accepted: *02 April 2015* Published: *24 April 2015*

#### Citation:

*Hayashi K, Kubo K, Kitazawa A and Nakajima K (2015) Cellular dynamics of neuronal migration in the hippocampus. Front. Neurosci. 9:135. doi: 10.3389/fnins.2015.00135* Keywords: hippocampus, migration, climbing mode, Ammon's horn, dentate gyrus, layer pattern

# Introduction

The hippocampal formation is a unique structure comprising the Ammon's horn (the hippocampus proper), dentate gyrus, entorhinal cortex, parasubiculum, presubiculum, and subicular complex. In the rodent brain, this architecture is located on and around the convexly curved medial lobule of the lateral cortex and is dorsally continuous with the neocortex. The hippocampus is a part of the limbic circuit and is functionally associated with spatial learning, as well as short- and long-term memory. In addition, functional magnetic resonance imaging (fMRI) analyses of some neuropsychiatric disorders have indicated its involvement in various types of mental activities; for example, decreased hippocampal volume was reported in patients with depression or post-traumatic stress disorder (PTSD) (Campbell et al., 2004; Woon et al., 2010). Anatomical abnormalities in the hippocampus are also observed in pathological conditions of some neuropsychiatric disorders, such as epilepsy, lissencephaly, and schizophrenia (Baulac et al., 1998; Harrison, 2004; Donmez et al., 2009). Some of these symptoms are thought to be associated with the migration deficit of hippocampal neurons during development (Barkovich et al., 1991; Dobyns et al., 1996; Montenegro et al., 2006). Neuronal migration in the neocortex is well-studied, and the cellular dynamics and molecular mechanisms involved in neuronal migration are also well-understood. Because the hippocampus and neocortex are included in the cerebral cortex, their neuronal migration was thought to be similar. However, differences in neuronal migratory behavior between these regions exist. Studies on cellular behavior of hippocampal neurons are broadly classified into two categories in terms of their methods, classical cellular labeling and molecular biological approaches. Classical techniques, such as Golgi staining and [3H] thymidine autoradiography labeling, were used to discover neuronal origins, cellular arrangements, neuronal migration paths, and neuronal morphologies in the hippocampus (Bayer, 1980; Nowakowski and Rakic, 1981; Rakic and Nowakowski, 1981; Altman and Bayer, 1990a,b,c). Furthermore, the development of molecular biological approaches, such as in utero electroporation, in utero virus transfer, and generation of transgenic mice, has shed light on the cellular dynamics of migrating neurons, successive behavior of neurons, neuronal lineages, and molecular mechanisms of hippocampal development (Nakahira and Yuasa, 2005; Li et al., 2009; Kitazawa et al., 2014; Seki et al., 2014; Xu et al., 2014).

In this review, we describe cellular dynamics and molecular mechanisms of migration of pyramidal neurons in the Ammon's horn and granule cells in the dentate gyrus during hippocampal development. There are three distinct hippocampal neuroepitheliums—the Ammonic neuroepithelium, the primary dentate neuroepithelium, and the fimbrial glioepithelium (Altman and Bayer, 1990a). Pyramidal neurons in the Ammon's horn are mainly generated from the Ammonic neuroepithelium and undergo radial migration to reach their final destination (Altman and Bayer, 1990b; Nakahira and Yuasa, 2005; Kitazawa et al., 2014), whereas cells comprising the dentate gyrus are originally produced from the primary dentate neuroepithelium (dentate notch), move in a migratory stream, and then migrate radially to form the dentate granule cell layer (Altman and Bayer, 1990a,c; Nakahira and Yuasa, 2005; Li et al., 2009; Seki, 2011; Li and Pleasure, 2014; Seki et al., 2014). We also compare neuronal migration between the neocortex and the hippocampus proper during development.

# Migration of Neocortical Pyramidal Neurons

Before describing migration of hippocampal neurons, we briefly outline migration of pyramidal neurons during rodent neocortical development (**Figure 1A**) to compare the migration mode between hippocampal pyramidal neurons and neocortical neurons (for a detailed illustration of neocortical neuronal migration, see reviews by Tabata et al., 2012; Evsyukova et al., 2013; Tan and Shi, 2013; Sekine et al., 2014). Pyramidal neurons generated in the neocortical ventricular zone (VZ) undergo morphological transformation before migrating up beneath the marginal zone (MZ), as summarized below.

Neocortical pyramidal neurons are generated from radial glial cells in the VZ (Miyata et al., 2001; Noctor et al., 2001) or from basal progenitors or basal radial glia in or around the subventricular zone (SVZ) (Noctor et al., 2004; Shitamukai et al., 2011; Wang et al., 2011). Neurons produced in the VZ remain there for at least 10 h with an apical process reaching the ventricular surface. The cells then move to just above the VZ (multipolar cell accumulation zone, MAZ), where they assume multipolar morphology and stay for about 1 day (Tabata and Nakajima, 2003; Tabata et al., 2009). Multipolar neurons in the MAZ repeatedly extend and retract multiple thin processes, and slowly wander and move toward the cortical plate (CP) (Tabata and Nakajima, 2003; Tabata et al., 2009). This unique behavior of multipolar neurons is called "multipolar migration." The multipolar neurons then transform into bipolar cells with a leading process extending

FIGURE 1 | Schematic diagrams of migration and layer arrangement on the neocortex and hippocampal CA1 during cortical development. (A) Neocortical neurons born between E10 and E12 radially migrate using the somal translocation mode. In contrast, late-born neurons transform their migration mode sequentially to multipolar migration, locomotion mode, and terminal translocation mode during their radial migration. These neurons form neocortical layers in a birthdate-dependent inside-out manner. (B) Hippocampal CA1 neurons born at late developmental stages change the migration mode to multipolar migration and then to the climbing mode. The migration mode used by early-born CA1 neurons remains unknown (somal translocation mode is a candidate). The layer arrangement in the Ammon's horn is thought to occur roughly in a birth-date dependent inside-out manner (another claim was also reported; see text for details). PP, preplate; VZ, ventricular zone; MZ, marginal zone; CP, cortical plate; IZ, intermediated zone; MAZ, multipolar cell accumulation zone; WM, white matter; HP, hippocampal plate; SLM, stratum lacunosum-moleculare; SR, stratum radiatum; SP, stratum pyramidale; SO, stratum oriens.

from a spindle-shaped cell body. These bipolar neurons migrate radially through the intermediated zone (IZ) and the CP along with a radial glial fiber. This migration mode is called "locomotion" (Rakic, 1972; Nadarajah et al., 2001). When the leading process of migratory neurons reaches the MZ, the neurons are thought to anchor the tip of the leading process in the MZ and leave from the radial glial fiber. Then, the neuronal cell body is pulled up while shortening the leading process and the cells stop just beneath the MZ (Nadarajah et al., 2001; Sekine et al., 2011). This final migration mode is termed "terminal translocation." Neurons born in the mouse VZ at E14, for example, take about 4–5 days to complete their migration (Ajioka and Nakajima, 2005). Because newly generated pyramidal neurons pass through earlier-born neurons before reaching beneath the MZ, pyramidal neurons are arranged in a birth-date-dependent inside-out manner, in which earlier-born neurons are positioned in the deep layers and later-born neurons are located in the more superficial layers in the CP (Angevine and Sidman, 1961).

# Migration of Hippocampal CA1 Pyramidal Neurons

The Ammon's horn is compartmentalized into the CA1, CA2, and CA3 along with the transverse axis, and horizontally divided Hayashi et al. Migration of hippocampal neurons

into several ordered layers—stratum oriens, stratum pyramidale (SP or the pyramidal cell layer), stratum radiatum (SR), and stratum lacunosum-moleculare (in only the CA3 region, the stratum lucidum exists between the SP and the SR). Pyramidal neurons in the hippocampal CA1 region are generated in the Ammonic neuroepithelium mainly from E16 to E20 with the peak around E18–19 in rats (Bayer, 1980; Altman and Bayer, 1990a,b), and from E12 to E18 with the peak between E14 and E16 in mice (Angevine, 1965; Caviness and Sidman, 1973; Smart, 1982; Kitazawa et al., 2014). Because the generation of pyramidal neurons and neuronal behaviors during development are different between the hippocampal CA1 and the CA3, we describe migratory behaviors of CA1 pyramidal neurons first and then those of CA3 neurons later (details of CA2 pyramidal neuronal migration are not well-known). Pyramidal neurons in the hippocampal CA1 region are mostly generated in the VZ, while a small population is produced from basal progenitors in the SVZ (Kitazawa et al., 2014). The newly born neurons leave the VZ and stay just above the VZ. These post-mitotic cells transform into multipolar cells with multiple thin processes and slowly move toward the IZ with repeated extension and retraction of these processes (Nakahira and Yuasa, 2005; Kitazawa et al., 2014). Existence of these multipolar neurons in the Ammon's horn has also been identified in the rabbit (Stensaas, 1967a,b) and the monkey (Nowakowski and Rakic, 1979). The time length of assuming multipolar morphology above the VZ (or in the hippocampal MAZ) differs depending on birthdates; neurons born at E12 or E13 stay as multipolar cells within 1 day, whereas those generated at E15 or E16 densely accumulate in the MAZ as multipolar cells for 3–4 days (Kitazawa et al., 2014). When late-born CA1 pyramidal neurons (generated from E14 to E16) move into the IZ, they transform into a bipolar spindle-shaped morphology, with one major leading process and multiple thin processes extending toward various directions (Nakahira and Yuasa, 2005; Kitazawa et al., 2014). These spindle-shaped neurons migrate through the IZ toward the hippocampal plate (HP, future stratum pyramidale), but sometimes transform their morphology back to a multipolar morphology. Upon observation of fixed tissue sections, the neurons seem to migrate along with radial glial fibers in the IZ, even when the fibers bend and curve (Nakahira and Yuasa, 2005). Recent time-lapse imaging has revealed that neurons in the IZ move obliquely at first and gradually migrate radially (Kitazawa et al., 2014), which coincides with the track of radial fibers. Nowakowski and Rakic also identified the apposition of neurons with radial glial fibers in the IZ of the monkey hippocampus in electron microscopic analyses (Nowakowski and Rakic, 1979). Just before neurons enter the HP, they extend one or two major branched leading process(es) with multiple thin processes (Nowakowski and Rakic, 1979; Kitazawa et al., 2014) and touch multiple radial glial fibers at the tip or the middle of the branched processes (Kitazawa et al., 2014). When they migrate through the HP, they dynamically extend and retract branched leading processes as if they were searching for the radial glial fibers. On the other hand, the cell soma of the migrating neuron moves up to the first branching point of the leading process. Subsequently, one of the branches grows and becomes a new leading process, followed by the movement of the cell soma again to the first branching point of the new leading process. In the HP of the CA1, migratory neurons repeat this process, thereby changing their migration scaffold (radial glial fiber) one after another until they reach the top of the HP. Consequently, hippocampal pyramidal neurons move in a zigzag manner, in contrast to the almost straight path of neocortical migrating neurons. Because this hippocampal migration mode is different from the wellknown modes of migration, it was termed a "climbing mode" (Kitazawa et al., 2014) (**Figure 1B**).

# The Difference between Migration of Hippocampal CA1 Pyramidal Neurons and that of Neocortical Neurons

There are a couple of similarities in the mode of neuronal migration between the hippocampal CA1 and the neocortex. One is the place of neuronal production, which is located in the VZ and SVZ in both structures. Neurons are generated near the ventricle and principally migrate toward the pial surface. The second similarity is the transformation into multipolar morphology when neurons migrate out of the VZ. The neurons reside in the MAZ for some time, and then transform into a bipolar morphology. However, there are also several major differences between the regions, such as migration mode, length of time required for completion of migration, and alignment pattern of the pyramidal neurons.

During development, neocortical neurons migrate in locomotion and terminal translocation modes through the CP (Nadarajah et al., 2001; Sekine et al., 2011; Evsyukova et al., 2013), while pyramidal neurons in the hippocampal CA1 region adopt a climbing migration mode, at least during the late stages of hippocampal development. Neocortical neurons in the locomotion mode migrate almost straight along individual radial glial fibers in the CP. In the outermost region of the CP [primitive cortical zone, PCZ (Sekine et al., 2011)], they take the terminal translocation mode before stopping beneath the MZ. In contrast, hippocampal neurons in the climbing migration mode migrate in a zigzag manner using several scaffold radial glial fibers in the HP (Kitazawa et al., 2014). The migratory speed for each migration mode is also different. The average migrating speeds of hippocampal CA1 neurons in the climbing mode and neocortical neurons in the locomotion mode are 7.1 and 20.5µm/h, respectively (Kitazawa et al., 2014). The speed of neocortical neurons in the terminal translocation mode is much faster, up to 50µm/1– 2 h (Sekine et al., 2011). Neocortical migrating neurons in a locomotion mode basically use a single radial glial fiber as the scaffold, whereas hippocampal neurons proceed using multiple radial glial fibers. The difference in these processes may bring about the difference in migration speed. Because cell density in the HP in late developmental stages is much greater than in the CP, with exception of the PCZ, it may be difficult for hippocampal CA1 neurons to migrate straight, unlike locomoting neurons in the neocortical CP. Because the Ammon's horn is widely extended during development (Altman and Bayer, 1990b), it is thought that pyramidal neurons born near the ventricle may need to move obliquely using the climbing mode of migration to fill up layers without gaps.

The second difference is the time spent migrating. Hippocampal CA1 pyramidal neurons generated at E15 or E16 spend 7–9 days to reach their final destinations (Tomita et al., 2011; Kitazawa et al., 2014), whereas it takes only 4–5 days for the neocortical late-born neurons to migrate beneath the MZ, although the migratory distance is much longer in the neocortex. This difference is observed not only in mice, but also in rats (Altman and Bayer, 1990b) and monkeys (Nowakowski and Rakic, 1981). Two factors apparently cause this difference. One is the difference in neuronal migration speed as mentioned above. The other is the time period when neurons remain in the MAZ as multipolar cells, termed "sojourning" cells by Altman and Bayer (1990b). Neocortical neurons spend about 1 day in the MAZ (Tabata et al., 2009). In contrast, the hippocampal CA1 neurons generated in late embryonic days spend almost 3 days in the MAZ, while this period varies depending on their birthdates (Nakahira and Yuasa, 2005; Kitazawa et al., 2014). Why do hippocampal neurons generated during late stages stay in the MAZ so long? There are at least two possibilities. One is the existence of alveolar channels, which are cell-free transient extracellular matrices dispersed in the IZ, just above the MAZ (Altman and Bayer, 1990b). Altman and Bayer displayed that this matrix becomes filled with axonal fibers of an unknown origin, and suggested that multipolar cells in the CA1 region might wait for the appearance of this matrix because of the connection to these axons (Altman and Bayer, 1990b). In addition, Deguchi et al. exhibited that neurons born with the same birthdate in the CA1, CA3, and dentate gyrus had similar gene expression patterns and preferentially connected with each other (Deguchi et al., 2011). Multipolar cells in the CA1 region may leave from the MAZ after CA3 neurons extend their axons and connect with them. In contrast, we showed that axon bundles appeared just above the MAZ at E15 and E16. These axon bundles originated from earlier-born neurons in the hippocampal CA1 region, and when they were transfected with GFP at E13.5, for example, the labeled axon bundles were located just above the multipolar cells at E18.5 (Kitazawa et al., 2014). The appearance of these axonal bundles coincided with the accumulation of multipolar cells in the hippocampal MAZ. CA1 neurons born on earlier days spend a much shorter time in a multipolar morphology and reach the pial surface in a short time, whereas axon bundles are not observed above multipolar cells in these earlier days (Kitazawa et al., 2014). Axonal bundles from earlier-born neurons may interfere with the migration of late-born pyramidal neurons. Future studies are needed to better understand the behavior of multipolar cells.

Finally, the pattern of neuronal alignment in the hippocampus may be different from the neocortex. Labeling experiments using [3H] thymidine indicate that hippocampal laminar formation occurs in a birthdate-dependent inside-out pattern in which earlier-born neurons comprise the deep SP region and later-born neurons join the superficial region, which is similar to the neocortical layer formation (Bayer, 1980; Rakic and Nowakowski, 1981; Altman and Bayer, 1990b). Recently, Xu et al. reported that hippocampal clonally related neurons in the CA1 region are distributed in a horizontal manner, not in a vertical column as neocortical neurons, shown using retroviral labeling and transgenic mice to label clonally related cells (Xu et al., 2014). The authors claimed that hippocampal layer formation did not occur in a birthdate-dependent inside-out pattern, contrary to previous reports. In addition to differences in their analytical method, this disparity might be explained as follows. Because neurons born on a certain day are distributed rather widely in the SP with a birthdate-dependent peak position, neurons with different birthdates are mixed with each other, obscuring the inside-out pattern. In addition to the birthdate-dependent positioning in the vertical/radial axis, however, it is also reasonable that the clonally related neurons are horizontally aligned. Because the SP (or HP) in the Ammon's horn region is horizontally expanded during development and is not as thick as the CP in the neocortex, this suggests the difficulty of clonally related neurons in the hippocampus proper to be aligned in a vertical manner like neocortical clonal neurons. Even if neurons in the HP of the Ammon's horn tend to align in an insideout pattern, they would also move to cover spaces in the layer yielded by the structural expansion, resulting in lateral/horizontal expansion of clonally related neurons. The climbing mode of migration is thought to be suitable to fill up gaps in the layer, because neurons in this mode can move to various directions, enabling clonal sister neurons to distribute broadly within the layer. Bending of radial glial fibers near the HP/SP would also be partly related to horizontal distribution of sister neurons (Xu et al., 2014), but similar bending of radial glial fibers beneath the CP/subplate is also observed in the neocortex, especially in the lateral part (Tabata and Nakajima, 2001), indicating that the bending morphology of radial fibers cannot fully explain the horizontal distribution of sister neurons in the hippocampus. Schematic models of neuronal behaviors in the neocortex and the Ammon's horn during development are illustrated in **Figure 1**.

# Migration of Hippocampal CA3 Pyramidal Neurons