REVIEW ARTICLE published: 22 January 2013 NEURAL CIRCUITS doi: 10.3389/fncir.2012.00123 The compartmental restriction of cerebellar

G. Giacomo Consalez 1 and Richard Hawkes 2*

1 Division of Neuroscience, San Raffaele Scientific Institute, Milan, Italy 2 Department of Cell Biology and Anatomy, Genes and Development Research Group, Faculty of Medicine, Hotchkiss Brain Institute, The University of Calgary, Calgary, AB, Canada

Edited by: The Purkinje cells (PC’s) of the cerebellar cortex are subdivided into multiple different Egidio D‘Angelo, University of Pavia, molecular phenotypes that form an elaborate array of parasagittal stripes. This array serves Italy as a scaffold around which afferent topography is organized. The ways in which cerebellar Reviewed by: interneurons may be restricted by this scaffolding are less well-understood. This review Leonard Maler, University of Ottawa, Canada begins with a brief survey of cerebellar topography. Next, it reviews the development Samuel S. Wang, Princeton of stripes in the with a particular emphasis on the embryological origins of University, USA cerebellar interneurons. These data serve as a foundation to discuss the hypothesis that *Correspondence: cerebellar compartment boundaries also restrict cerebellar interneurons, both excitatory Richard Hawkes, Department of Cell [granule cells, unipolar brush cells (UBCs)] and inhibitory (e.g., Golgi cells, basket cells). Biology and Anatomy, Genes and Development Research Group, Finally, it is proposed that the same PC scaffold that restricts afferent terminal fields to Faculty of Medicine, Hotchkiss Brain stripes may also act to organize cerebellar interneurons. Institute, University of Calgary, 3330 Hospital Drive N.W., Calgary, Keywords: , stripe, zone, , , , unipolar brush cell, AB T2N 4N1, Canada. e-mail: [email protected]

REVIEW OF CEREBELLAR COMPARTMENTATION shock protein (HSP)25: Armstrong et al. (2000)], the L7/pcp2 The architecture of the adult cerebellar cortex is built around hun- transgene (Oberdick et al., 1993; Ozol et al., 1999)andhuman dreds of modules (“stripes”), each comprising no more than a natural killer cell antigen 1 (HNK1: Eisenman and Hawkes, 1993; few hundred Purkinje cells (PC’s: Hawkes et al., 1997; Apps and Marzban et al., 2004 identify subsets of zebrin II+ PCs.). The Hawkes, 2009: Figure 1). Along the rostrocaudal axis, the cere- pattern of zones and stripes is symmetrical about the midline, bellar cortex is divided into five transverse zones—the anterior highly reproducible between individuals and insensitive to exper- zone (AZ: ∼lobules I–V), central zone anterior (CZa: ∼VI), cen- imental manipulation [see below, and reviewed in Larouche and tral zone posterior (CZp: ∼VII), posterior zone (PZ: ∼VIII–IX), Hawkes (2006); Apps and Hawkes (2009)]. The implication is that and nodular zone (NZ: ∼X). Transverse zones and zonal bound- the adult cerebellar cortex of the mouse is highly reproducibly aries are revealed by expression patterns (e.g., Odutola, 1970; subdivided into several hundred distinct stripes with >10 dis- Prasadarao et al., 1990; Eisenman and Hawkes, 1993; Millen et al., tinct PC molecular phenotypes (Hawkes, 1997; Apps and Hawkes, 1995; Alam et al., 1996; Ozol et al., 1999; Armstrong et al., 2000; 2009). Eisenman, 2000; Logan et al., 2002; Marzban et al., 2008; etc.), Transverse zones and parasagittal stripes are important reflect patterns of cell death in many genetic mutations or toxic because cerebellar patterning influences all aspects of cerebel- insults [reviewed in Sarna and Hawkes (2003)], and coincide lar organization and function. The most-studied example is that with boundaries in the actions of mutations that disrupt cere- the terminal fields of both climbing fibers and mossy fibers bellar development and structure (Herrup and Wilczynsk, 1982; are aligned parasagittally with stripes of PCs (climbing fibers: Hess and Wilson, 1991; Napieralski and Eisenman, 1993, 1996; Gravel et al., 1987; Voogd and Ruigrok, 2004; Sugihara and Quy, Ackerman et al., 1997; Armstrong and Hawkes, 2001; Beirebach 2007; etc.; mossy fibers: Gravel and Hawkes, 1990; Akintunde et al., 2001; etc.). and Eisenman, 1994; Ji and Hawkes, 1994; Sillitoe et al., 2003; Each transverse zone is further subdivided from medial to lat- Armstrong et al., 2009; Gebre et al., 2012; etc.). eral into stripes. For example, Figure 1 shows alternating zones The molecular topography of the cerebellar cortex correlates and stripes in cerebella immunostained for zebrin II (Brochu nicely with the functional maps [see Apps and Garwicz (2005); et al., 1990 = aldolase C (Aldoc)—Ahn et al., 1994; Hawkes Apps and Hawkes (2009)]. For example, mossy fiber tactile recep- and Herrup, 1996; Sillitoe and Hawkes, 2002) and phospholi- tive field boundaries correlate well with zebrin II+/− stripe pase C (PLC)β4—Sarna et al., 2006). Many molecular markers boundaries [Chockkan and Hawkes, 1994; Hallem et al., 1999: co-localize with either the zebrin II+ or PLCβ4+ stripes [e.g., see also Chen et al. (1996)]. More recently, Wylie et al. have reviewed in Sillitoe et al. (2011); Sillitoe and Hawkes (2013)]. demonstrated an elegant correlation between PC stripes and Furthermore, other markers reveal subdivisions within stripes complex spike activity boundaries associated with optic flow in (e.g., the patterns of afferent terminal fields: Akintunde and the pigeon vestibular zone (e.g., Graham and Wylie, 2012). The Eisenman, 1994; Ji and Hawkes, 1994, 1995; etc.) and addi- reproducible association of function with specific stripes also tional PC subtypes within the zebrin II+/− families [e.g., heat presents a potential substrate for function-specific adaptations at

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FIGURE 1 | An overview of cerebellar compartmentation. Whole and paraflocculus; l. ans (c1 and c2), lobulus ansiformis, crus 1 and crus 2; mount views of the complementary stripe arrays revealed by l. para/pm, lobulus paramedianus; SL, lobulus simplex; AZ, anterior zone; CZ, immunoperoxidase-staining for phospholipase cβ4(PLCβ4) and zebrin central zone; PZ, posterior zone; NZ, nodular zone; lobules are indicated by II/aldolase C in the adult mouse. (Views: above—anterior; middle—dorsal; Roman numerals (I–X). Scale bar is in mm [Adapted from Sarna et al. (2006) lower—posterior). Abbreviations: l. sim, lobulus simplex; floc/PFI, flocculus and Sillitoe and Hawkes (2002)]. the molecular level. For instance, many of the molecules thought Because we argue that much cerebellar patterning is built to mediate synaptic transmission and long-term depression at around a PC zone and stripe scaffold, we begin with a brief the parallel fiber-PC synapse show stripe restriction [including review of the origins of PC zones and stripes [reviewed in Herrup metabotropic glutamate receptors (Mateos et al., 2001), excita- and Kuemerle (1997);ArmstrongandHawkes(2000);Larouche tory amino acid transporter 4 (Dehnes et al., 1998), PLC (Tanaka and Hawkes (2006); Sillitoe and Joyner (2007); Apps and Hawkes and Kondo, 1994; Sarna et al., 2006), protein kinase C (Chen (2009); Dastjerdi et al. (2012); Sillitoe and Hawkes (2013)]. The and Hillman, 1993; Barmack et al., 2000), neuroplastin (Marzban cerebellar primordium arises from the rostral et al., 2003), GABA receptors (Chung et al., 2008a), and so between E8.5 and E9.5 (e.g., Wang et al., 2005; Sillitoe and Joyner, on]. Consistent with this hypothesis, electrophysiological studies 2007: all timings are for mice). It houses two distinct germinal have confirmed differences in parallel fiber-PC synaptic behavior matrices—the dorsal (RL) and the ventral ventricu- between stripes (e.g., Wadiche and Jahr, 2005; Paukert et al., 2010; lar zone (VZ) of the 4th ventricle. Genetic fate mapping shows Ebner et al., 2012). that a Ptf1a expressing domain in the VZ gives rise to all PCs Thus, both patterns of gene expression and functional maps (Hoshino et al., 2005; Hoshino, 2006). The Ptf1a+ VZ is not in the cerebellum seem to share a common architecture. The homogenous and gene expression differences further subdivide it present review considers some of the evidence that PC stripe (including Ascl1, Neurogenin 1/2, Lhx1/5, etc.—Chizhikov et al., architecture also restricts the distributions of cerebellar interneu- 2006; Salsano et al., 2007; Zordan et al., 2008). PCs undergo ter- rons. We begin with an overview of cerebellar pattern formation minal mitosis in the VZ between E10 and E13 (Miale and Sidman, during development, then discuss the origins and development 1961). of the various cerebellar interneurons, review the evidence that Adult PC zebrin II+/− phenotypes are specified early in interneurons are restricted to particular zones and stripes, and development and birthdating studies in mice have identified two conclude by proposing the general hypothesis that interactions PC populations—an early born subset (E10–E11.5) mostly des- between interneurons and PCs during development are an impor- tined to become zebrin II+ and late-born subset (E11.5–E13) tant mechanism that restrict distributions. destined to become zebrin II—(Hashimoto and Mikoshiba, 2003;

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Larouche and Hawkes, 2006; Namba et al., 2011). Many exper- etc. [see Schilling et al. (2008)]—but nothing is known of their imental interventions—in vitro culture models, cerebellar trans- patterns of restriction and they will not be considered further plants, afferent lesions, sensory deprivation, etc.—have been used below. to try to alter adult PC zebrin II+/− phenotypes, but these have always proved ineffective [reviewed in Larouche and Hawkes THE EMBRYOLOGICAL ORIGINS OF CEREBELLAR (2006)]. In fact, the only experimental manipulation known to INTERNEURONS alter PC subtype identity is deletion of the atypical helix-loop- Upon completion of early cerebellar patterning, neurogenesis helix transcription factor Early B-cell Factor 2 (Ebf2), a repressor begins. Two germinative compartments are established, the VZ of the zebrin II+ phenotype (Croci et al., 2006; Chung et al., and the rostral RL. In the mouse, this second phase of cere- 2008b). bellar development starts between E9 and E11 and proceeds Postmitotic PCs migrate out of the VZ and stack in the for many days, giving rise to the different classes of cerebellar cortical transitory zone with the earliest-born located dorsally cells (Figure 2). At the onset of neurogenesis, the cerebellar pri- and the youngest ventrally. Subsequently, the PCs reorganize to mordium consists of two symmetric bulges extending dorsally yield a stereotyped array of embryonic clusters with multiple and laterally from the midline of rhombomere one. These two molecular phenotypes [E14–E18: reviewed in e.g., Herrup and halves are fated to eventually fuse at the midline, giving rise to a Kuemerle (1997)]. Starting at around E18, the embryonic clusters single dorsal formation spanning, and eventually exceeding, the disperse, triggered by Reelin/Disabled-1 (Dab1) signaling (e.g., width of the 4th ventricle. The inner and outer germinal layers Armstrong and Hawkes, 2000; Larouche and Hawkes, 2006; Apps of the cerebellar plate constitute the VZ and the RL, respectively and Hawkes, 2009). As the clusters disperse into adult stripes the (Altman and Bayer, 1997). PCs spread to form a monolayer. Because dispersal occurs primar- A series of studies conducted since 1990 have unveiled the ori- ily in the anteroposterior plane, the clusters string out into long gin of GABAergic and glutamatergic that populate the parasagittal stripes (e.g., Marzban et al., 2007). cerebellar primordium and, eventually, the adult cerebellar cor- The embryonic PC clusters are the targets for ingrowing tex. The development of RL-derived progenitors is affected by climbing and mossy fiber afferents. Climbing fibers from the signals produced by the roof plate (Alder et al., 1999; Millonig contralateral inferior olive enter the cerebellar cortex prenatally et al., 2000; Chizhikov et al., 2006). These progenitors soon (Sotelo, 2004), and contact with PC clusters can be identified become positive for the proneural gene Atoh1/Math1 (Machold from birth (e.g., Mason et al., 1990).ItappearsthatasthePCclus- and Fishell, 2005; Wang et al., 2005). Targeted disruption of Atoh1 ters disperse into parasagittal stripes the climbing fiber terminal virtually ablates the entire repertoire of cerebellar glutamater- fields ride along with them, thereby maintaining the embryonic gic neurons (Jensen et al., 2004; Wang et al., 2005). Progenitor topographical relationship and assuring a reproducible coupling cells originating in the RL first migrate tangentially, dispersing between specific subnuclei of the inferior olivary complex and over the dorsal surface of the cerebellar primordium, and then specific PC stripes [reviewed in Ruigrok (2011)]. Postnatally, move radially into the cortex or cerebellar nuclei (CN). The first extensive pruning of the climbing fiber projection occurs until cells to migrate tangentially from the RL (circa E10.5) give rise each PC receives input from only one cell in the inferior olive, but this does not seem to contribute significantly to the refine- ment of the topography (Crépel, 1982). A similar sequence of events also patterns the mossy fiber projections, which are found in direct association with embryonic PC clusters from (circa E15: Grishkat and Eisenman, 1995; and possibly earlier—e.g., Morris et al., 1988). In the adult cerebellar cortex mossy fibers do not directly contact PCs. Rather, between P0 and P20, as the granu- lar layer matures, mossy fiber afferents detach from the PCs and form new synapses with local granule cells. As a result, mossy fiber terminal fields retain their alignment with the overlying PC stripes (e.g., Gravel and Hawkes, 1990; Matsushita et al., 1991; Akintunde and Eisenman, 1994; Ji and Hawkes, 1994, 1995; Apps and Hawkes, 2009). FIGURE 2 | Temporal sequence of birth for the main types of neurons The aim of this review is to assess the evidence first that cere- that populate the adult cerebellum. Projection neurons are in boldface, bellar interneurons show restriction and secondly to review the interneurons are in normal text. Projection neurons of the cerebellar nuclei hypothesis that the PC architecture is the template around which and cortex are the first to be born at the outset of cerebellar neurogenesis. they organize. This is a straightforward extension of the model These include glutamatergic nuclear projection neurons (NP) derived from the rhombic lip (RL) and GABAergic nucleo-olivary projection neurons (NO) previously espoused for the development of cerebellar afferent and Purkinje cells (PC) derived from the ventricular zone (VZ). Local topography (e.g., Sotelo, 2004). The main classes of cerebellar interneurons (of both neurotransmitter phenotypes) are born during late interneurons are granule cells and unipolar brush cells (UBCs; embryonic and early postnatal development. GABAergic interneurons are glutamatergic—excitatory), and Golgi, stellate, and basket cells generated according to an inside-out sequence, occupying the deep nuclei (GABAergic—inhibitory). In addition, there are several other first, and then the granular and molecular layer [Modified from Carletti and Rossi (2008)]. types of inhibitory interneuron—Lugaro cells, Chandelier cells,

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to glutamatergic projection neurons of the CN (Machold and et al., 2011). Precursors expressing neurogenin 1 (Neurog1) Fishell, 2005). They enter the primordium from just below its become PCs or cortical GABA interneurons (Kim et al., 2008; surface, giving rise to a transient structure sometimes referred Lundell et al., 2009). Neurog2+ precursors give rise to PCs and to as nuclear transitory zone (NTZ), which will evolve into GABAergic CN neurons, including presumptive nucleo-olivary the CN. Shortly thereafter (circa E11–E14), a second echelon neurons and interneurons (Florio et al., 2012). Two subsets orig- of RL-derived glutamatergic progenitors disperses by tangential inate from that population. The first, located anteriorly and migration to populate the external granular layer (EGL). These medially, starts expressing the proneural gene Neurog1 and gives cells are fated to give rise to granule cell neurons only (Hallonet rise to cortical interneurons of the GL (Golgi, Lugaro) and ML et al., 1990; Alvarez Otero et al., 1993). Starting shortly before (basket, stellate) (Lundell et al., 2009; Kim et al., 2011). The birth, these progenitors undertake a long phase of clonal expan- second group, positive for Neurog2, gives rise to CN interneu- sion in the EGL (between E17 and P20), under control of signals rons (Florio et al., 2012). While the cell surface marker Neph3 secreted by PCs (Smeyne et al., 1995; Dahmane and Ruiz-i-Altaba, is expressed throughout the VZ, including interneuron progen- 1999; Wallace, 1999; Wechsler-Reya and Scott, 1999; Lewis et al., itors, E-cadherin (Cdh1) is differentially expressed, with higher 2004). levels found on the surface of mitotic PC progenitors (Mizuhara In addition to CN neurons and GCs, a third population of et al., 2009). To date, it is not clear if some progenitors co-express glutamatergic neurons originates in the embryonic cerebellum Neurog1 and Neurog2. between E15 and E17: the so-called UBCs. UBCs are glutamater- gic interneurons of the granular layer, with small somata, mossy ZONE AND STRIPE BOUNDARIES RESTRICT CEREBELLAR fiber-like axon terminals, and brush-like dendrites (Altman and INTERNEURONS Bayer, 1977; Mugnaini and Floris, 1994; Diño et al., 1999, 2000, GRANULE CELLS 2001; Nunzi and Mugnaini, 2000; Nunzi et al., 2001, 2002). Like The most plentiful cerebellar interneuron is the granule cell, granule cell progenitors, UBCs originate in the RL (Englund et al., which comprises almost all the neurons of the cerebellum. 2006), and migrate inwards to invade the of prospec- Granule cells receive their input from mossy fibers (mostly tive lobule X. From there they disperse, populating the granular directly but in some cases via UBCs), and synapse in the molecu- layer and extending glutamatergic axons akin to mossy fibers to lar layer as parallel fiber synapses on PC dendrites and inhibitory form synapses with granule cell dendrites. interneurons. The development of granule cells has been stud- Unlike glutamatergic neurons, all GABAergic neurons of the ied extensively [e.g., reviewed in Chédotal (2010); Butts et al. cerebellum originate in a ventral germinative epithelium lining (2011); Hashimoto and Hibi (2012)]. After several proliferative the 4th ventricle, called the VZ and recent evidence indicates cycles, granule cell precursors located in the outer part of the that, as for granule cell proliferation, VZ progenitor prolifera- EGL exit the cell cycle, express differentiation markers, and mod- tion is also controlled by sonic hedgehog (Huang et al., 2010). ify their repertoire of adhesion molecules (e.g., Xenaki et al., Projection neurons are generated first and local interneurons are 2011). Through these surface molecules, in particular astrotactin born during late embryonic and early postnatal life (Miale and (Edmondson et al., 1988), they contact the distal processes of Sidman, 1961; Pierce, 1975; Altman and Bayer, 1997; Morales Bergmann glia and undertake a centripetal radial migration in and Hatten, 2006). While GABAergic projection neurons only the course of which they begin to populate the granular layer, proliferate in the VZ, interneurons derive from progenitors that migrating inwards across the PC layer while extending T-shaped delaminate from the VZ into the prospective white matter. axons (future parallel fibers) orthogonal to their migration path Projection neurons (PCs and nucleo-olivary neurons) prolifer- (Altman and Bayer, 1997). Upon their arrival in the granular ate in the VZ and become committed to their fate at early layer, granule cells receive inputs from mossy fiber presynap- stages of development, acquiring their mature subtype pheno- tic terminals, and trans-synaptically induce their maturation by types through cell-autonomous mechanisms [for example, see secreting Wnt family ligands (Hall et al., 2000). Florio et al. (2012)]. Conversely, interneurons derive from pro- genitors that delaminate into the prospective white matter, where Restriction they develop in an inside-out progression (CN to granular layer Several lines of evidence point to an elaborate parcellation of the to molecular layer) from a single pool of progenitors. While granular layer, with evidence of restriction both into transverse sojourning in the white matter, their fate choices, production zones and parasagittal stripes. Differences in gene expression rates, and differentiation schedules remain flexible and are largely have revealed multiple granule cell subtypes [e.g., Otx1/2— dependent on stage-specific extracellular cues (Leto et al., 2006, Frantz et al., 1994; nicotinamide adenine dinucleotide phosphate 2009). (NADPH)-oxidase—Hawkes and Turner, 1994;fibroblastgrowth In regard to gene expression, all VZ-derived progenitors factor (FGF)1—Alam et al., 1996; Eph receptors and Ephrins— express Ptf1a, a gene encoding a bHLH transcription factor, as Rogers et al., 1999, etc.; reviewed in Hawkes and Eisenman shown by targeted inactivation studies (Hoshino et al., 2005; (1997); Ozol and Hawkes (1997)]. Multiple granule cell sub- Pascual et al., 2007). Ptf1a+ progenitors start regulatory cascades types can be explained in two broad ways: either they repre- leading to the expression of other proneural genes (Zordan et al., sent granule cell lineages or they are a secondary response to 2008; Dastjerdi et al., 2012; Consalez et al., 2013). Cerebellar their local environment (for example, the local mossy fibers or Ascl1/Mash1+ precursors give rise to PCs and to all GABAergic PCs). In many cases, we cannot distinguish these possibilities. interneurons (Kim et al., 2008; Grimaldi et al., 2009; Sudarov However,theanalysisofmurineembryonicstemcellchimeras

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the PCs, and it is therefore noteworthy that the granular layer lineage restriction boundaries roughly align with PC transverse zone boundaries, and suggests that distinct PC compartments may direct the spreading EGL into distinct migratory streams. Consequently, as the EGL comes to cover the cerebellar sur- face, granule cell lineage discontinuities end up aligned with the PC transverse zone boundaries. Subsequently, both intrin- sic differences between granule cell populations and epigenetic interactions between developing granule cells and PCs could contribute to selective patterns of granule cell gene expression. Notably, PCs express extracellular factors during early postnatal development. One of them, Igf-1, is expressed in a pattern very FIGURE 3 | Compartmentation of the granular layer. Cell transverse lineage boundaries seen in a β-gal-stained sagittal section through an adult similar to the distribution of zebrin II—PC stripes. Igf-1 acts in murine embryonic stem cell chimera. The ES-cell-derived granule cells an autocrine/paracrine fashion to protect PCs from apoptotic cell (β-gal+) are concentrated preferentially in the anterior vermis (AZ) with a death, particularly at birth, and its expression is driven locally restriction boundary in lobule VI (AX/CZ: arrow), and in the nodulus with a by EBF2 expressed by PCs (Croci et al., 2011). Abundant evi- boundary in the sulcus between lobules IX and X (the PZ/NZ boundary: dence supports a role for Insulin-like growth factor 1 and 2 (IGF1 arrow) [Adapted from Hawkes et al. (1999)]. and IGF2) in central nervous system (CNS) development. IGF1 is predominantly expressed in neurons in a fashion that coin- cides with outbursts of neural progenitor proliferation, neurite has revealed two consistent granule cell lineage boundaries, one outgrowth, and synaptogenesis (D’Ercole et al., 1996; Bondy and located close to the AZ/CZ PC boundary and a second to the Cheng, 2004; Ozdinler and Macklis, 2006; Fernandez and Torres- PZ/NZ boundary (Hawkes et al., 1999: Figure 3). An AZ/CZ Alemàn, 2012). A recent paper indicates that Igf-1, whose levels granule cell boundary can also be seen in the granular layer of fluctuate with light-dark cycles, promotes granule cell migration several mouse mutants (e.g., scrambler—Goldowitz et al., 1997; into the GL (Li et al., 2012). Thus, it is conceivable that gran- disabled—Gallagher et al., 1998), and studies with weaver (wv/wv) ule cells in contact with Igf1-expressing (zebrin II-) PCs may X +/+ and M. musculus X M. caroli chimeras also revealed devel- migrate faster into the GL and thus establish privileged synaptic opmental boundaries at these sites (Goldowitz, 1989). Evidence contacts. for a distinct AZ compartment in the EGL also comes from anal- Beyond the restriction of granule cell subtypes to transverse ysis of Unc5h3 X +/+ (Goldowitz et al., 2000)and(smalleye) zones, several granule cell markers reveal a much more elaborate Sey/SeyNeu X +/+ chimeras (Swanson and Goldowitz, 2011), and parcellation into parasagittal stripes [e.g., in the expression pat- from the effects of several mouse mutations (e.g., rostral cerebel- terns of acetylcholinesterase (Marani and Voogd, 1977; Boegman lar malformation—Eisenman and Brothers, 1998; NeuroD−/−— et al., 1988), cytochrome oxidase (Hess and Voogd, 1986; Leclerc Miyata et al., 1999). Finally, patterns of granular layer and/or et al., 1990), and nNOS (Yan et al., 1993; Hawkes and Turner, EGL gene expression show the AZ/CZ (e.g., acidic FGF, receptor 1994; Schilling et al., 1994)]. It is plausible that this molecular protein tyrosine phosphatase—McAndrew et al., 1998; Otx-1— complexity is related to the complex array of somatotopic patches Frantz et al., 1994)andPZ/NZ(e.g.,Otx1—Frantz et al., 1994; mapped in some cerebellar regions (Welker, 1987; Hallem et al., En2—Millen et al., 1995; Tlx3—Logan et al., 2002; Lmx1a+— 1999; Apps and Hawkes, 2009). Finally, perhaps the most curious Chizhikov et al., 2010) boundaries. Similarly, neuronal nitric manifestation of granular layer heterogeneity is the reproducible oxidesynthase(nNOS:NADPH)isexpressedbymostgran- array of wrinkles in the granular layer (“blebs”) that are seen ule cells but is entirely absent from those of the NZ (Hawkes when ethanol-fixed, paraffin-embedded sections are rehydrated and Turner, 1994). While epigenetic interactions with PCs may (Hawkes et al., 1997, 1998). The structural basis of blebbing is not explain differential gene expression, they cannot account for the known, but it points to the possibility that blebs represent indi- spatial distribution of genotypes in the chimeras. We therefore vidual cytoarchitectonic units and that the mouse granular layer conclude that the cerebellar granular layer has multiple lineage is subdivided into several thousand modules [reviewed in Hawkes histories and derives from multiple distinct precursor pools either (1997)]. side of lineage boundaries within the RL. It has been established Do these granular layer stripes arise through lineage restric- by genetic fate mapping that early born granule cell progenitors tion or are they secondary responses to the local environment (E12.5–E15.5), migrate preferentially into the anterior vermis, (e.g., the type of mossy fiber input or the local PCs)? It is not whereas later born ones distribute more evenly along the AP axis, known but it is difficult to imagine a mechanism by which gran- and only late-born ones (circa E17) populate lobule X (Machold ule cell stripes form through the targeted migration of granule cell and Fishell, 2005). subtypes to hundreds of destinations (although raphes between What determines the location of the granular layer lineage PC clusters do seem to preferentially guide the descent of imma- boundaries? While some signals may be intrinsic to early born ture granule cells to the granular layer: e.g., Karam et al., 2000; granule cell progenitors in the RL, the most obvious source Luckner et al., 2001), so it is more plausible that stripe molecu- of positional information for the developing granular layer (or, lar phenotypes among granule cells are secondary responses to more likely, the developing EGL) is the compartmentation of local cues. One mechanism might be that granule cells adopt

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their molecular phenotypes according to the local PC subtype AZ, and the PLCβ4+ subset is very rare in the AZ (Mugnaini and environment through which they migrate (and synapse) dur- Floris, 1994; Diño et al., 2000; Chung et al., 2009a). Each sub- ing postnatal development (several studies have demonstrated set is also loosely restricted to stripes that align with PC stripes PC influences on granule cell growth and differentiation—e.g., (e.g., Chung et al., 2009b: Figure 4). This distribution implies that PC-derived sonic hedgehog regulates granule cell proliferation each UBC subset receives afferent input from a different set of (Wallace, 1999); PC-derived brain-derived neurotrophic factor mossy fiber inputs. However, it seems unlikely that UBC subtype (BDNF) stimulates granule cell migration—Borghenasi et al., phenotypes are secondary to mossy fiber input since when they 2002). An alternative mechanism might be that granule cell sub- are allowed to mature as dissociated cells in vitro,intheabsence types are specified by the type of mossy fiber (or UBC) afferent of extracerebellar mossy fiber cues, the different phenotypes are input they receive. As noted earlier (section “Review of cerebel- all expressed (Anelli and Mugnaini, 2001; Chung et al., 2009a). lar compartmentation”) mossy fiber terminal fields from different It is therefore plausible that the restricted distributions of UBC sources and of different molecular phenotypes are restricted to parasagittal stripes that align with PC stripes (e.g., somatostatin- immunoreactive—Armstrong et al., 2009; vesicular glutamate transporter immunoreactive—Gebre et al., 2012). Perhaps differ- ential mossy fiber innervation specifies granule cell subtype. In both cases, PC specification or mossy fiber specification of gran- ule cell subtype, granule cell stripes would naturally also align with PC stripes. This is consistent with the demonstration by Schilling et al. (1994) that ingrowing mossy fibers may downregulate nitric oxide synthase expression and thereby contribute to the generation of granule cell subtypes.

UNIPOLAR BRUSH CELLS UBCs are glutamatergic interneurons of the granular layer. They receive mossy fiber innervation, in large part from primary vestibular afferents (e.g., Diño et al., 2000, 2001), and project in turn to granule cell dendrites. At least three subtypes of UBC are known, one immunoreactive for calretinin (= CR+ subset), another expressing both the metabotropic glutamate receptor (mGluR1α: Nunzi et al., 2001, 2002)andPLCβ4(Chung et al., 2009a = the mGluR1α+ subset), and a third expresses PLCβ4but not mGluR1α (Chung et al., 2009a = the PLCβ4+ subset). UBCs are born between E15 and P2 (Abbott and Jacobowitz, 1995; Sekerková et al., 2004; Chung et al., 2009b). They appear to have two distinct origins. The majority arise ventrally, possi- bly in the VZ of the fourth ventricle (e.g., Ilijic et al., 2005)but more likely from the RL since RL ablation in slice cultures signif- icantly reduced the number of UBCs (Englund et al., 2006)and theproductionofUBCsisdecreasedintheMath1 null cerebel- lum (as mentioned, Math1 is required for the development of RL derivatives: Machold and Fishell, 2005; Wang et al., 2005). Either way, most UBCs migrate into the developing cerebellar anlage soon after the PCs arrive (E14 onwards: Abbott and Jacobowitz, 1995) and then disperse via the white matter tracts, presumably FIGURE 4 | Unipolar brush cells are restricted at stripe boundaries in guided by cues associated with PC axons or afferent projections. the adult mouse cerebellum. (A) Cerebellar stripe topography is built In addition, a second, small population of UBCs arises dorsally around PC subtypes. Immunoperoxidase staining of a transverse section from the EGL (and presumably the RL) and reaches the gran- through lobule IX for zebrin II reveals three broad stripes of immunoreactive ular layer by following the same dorsoventral migratory route PCs [P1+ at the midline, P2+ and P3+ laterally on either side: for stripe + as the granule cells (Abbott and Jacobowitz, 1995; Chung et al., nomenclature, see Sillitoe and Hawkes (2002)]. (B) CR UBC clusters (red) in lobule IX align with the zebrin II P1+ and P3+ PC stripes (green). 2009b). (C) mGluR1α+ UBCs (green) in lobule IX cluster beneath the midline P1+ Each UBC subset has a characteristic topographical distribu- PC stripe (red). (D) Double immunostaining with anti-PLCβ4 (red) and tion (Braak and Braak, 1993; Floris et al., 1994; Diño et al., 1999; anti-zebrin II (green) shows that PLCβ4-immunopositive UBCs are uniformly Nunzi et al., 2002; Chung et al., 2008a): all three are concentrated distributed in cerebellar lobule IX (the combined PLCβ4+ and mGluR1α+ = μ preferentially in the NZ, but mGluRlα+ UBCs are also common subsets). Scale bars: D 125 m (A–D) [Adapted from Chung et al. (2009b)]. throughout the vermis, only occasional CR+ UBCs are seen in the

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subtypes comes about because each uses different topographical layer (basket) and less so for the more superficial ones (stellate), cues to guide their migrations. Experiments using an Ebf2−/− both the axons and the dendrites tend to be oriented parasagit- mouse support this inference. When Ebf2 is deleted, many PCs tally. For example, a classic basket cell contacts about 40 PCs. express abnormal molecular phenotypes (a mixture of zebrin Their terminal fields are ovoid in shape, four times longer than II+ and zebrin II−: Croci et al., 2006; Chung et al., 2008b). they are wide, and with their long axes aligned parasagittally with Notably, anterior vermis PCs adopt features of the posterior ver- the PC stripes (e.g., Eccles et al., 1967; Rakic, 1972; King et al., mis. Interestingly, in these mice, the normal restriction of UBCs 1993). This axial ratio is much less for stellate cells. Ever since to the posterior vermisis alsolost, and UBC profilesbecome plen- the work of Szentagothai (1965) it has been recognized that the tiful in the anterior lobules (Chung et al., 2009b). Because EBF2 is parasagittal orientation of basket cells represents a substrate for not expressed by UBCs this suggests that the abnormal topogra- PC lateral inhibition. More recently, and consistent with the mor- phy in the mutant is not cell-autonomous but rather secondary to phology, physiological studies both in vitro and in vivo confirm the abnormal PC transdifferentiated phenotype. This is also con- that the inhibitory fields of basket/stellate cells are confined to a sistent with the developmental data showing a close association single stripe, with molecular layer inhibition restricted parasagit- between PCs and UBCs in the perinatal cerebellum (Chung et al., tally (Ekerot and Jörntell, 2001, 2003; Jörntell and Ekerot, 2002; 2009b). Finally, mutations in the Reelin signaling pathway result Gao et al., 2006; Dizon and Khodakhah, 2011;etc.:thefunc- in a failure of PC cluster dispersal. This results in a correspond- tional implications of reciprocal inhibition between interneurons ing UBC ectopia. For example, in scrambler (a Disabled1 mutant: within a microzone have recently been reviewed—Jörntell et al., Sheldon et al., 1997) UBCs are found in association with specific 2010). PC ectopic clusters (Chung et al., 2009b). Taken together, the data How do basket/stellate cells acquire their parasagittal orien- support the hypothesis that PCs present topographically orga- tations? First, they are born too late to interact with embryonic nized cues to the growth cones of migrating UBCs and thereby PC clusters (from P2 to P19: and anyway there is little evidence restrict their topography. of subtype specification). However, the parasagittal orientation of basket cell axonal arbors can still be explained by PC rostrocau- BASKET AND STELLATE CELLS dal spreading. Molecular layer interneurons invade the immature Basket and stellate cells are small inhibitory interneurons of the molecular layer randomly from the white matter. Once in the molecular layer. Whether or not they represent two distinct cell molecular layer they contact a local cluster of some 40 PCs. In classes or a morphological continuum is unclear (e.g., Schilling the course of the next 3 weeks, these PCs gradually disperse et al., 2008): for our purposes we will discuss them together. rostrocaudally, so that the cerebellar cortex extends more than There is little evidence of distinct subclasses of basket/stellate 10-fold in rostrocaudal extent with almost no change in width. cells (cyclin D2 expression can distinguish subtypes, but this is As a result, the basket/stellate cell terminal field becomes a short, amenable to other explanations: Huard et al., 1999). An excep- parasagittal PC stripe (Figure 5). It is not clear to what extent the tion is the study of Chen and Hillman (1993)whoshowedthat basket/stellate terminal fields are restricted to particular stripes. It aproteinkinaseCδ-immunoreactive basket/stellate cell subset in could be that there are as yet unrecognized subtypes (as for UBCs, the rat cerebellum is strongly concentrated in the AZ. for example), or that secondary pruning refines their arbors, or All GABA interneuron progenitors transiently activate Pax2 the restriction could be purely statistical. In any case, PC dispersal expression around cell cycle exit. Before homing in on their final would result in a continuum of terminal field shapes: the earliest- location, the young Pax2+ interneurons reside for several days born interneurons enter the molecular layer first and therefore in the white matter, progressing in their maturation, and acquir- develop the most extended parasagittal terminal fields (basket ing their final identities. Postmitotic Pax2+ neurons harvested cell); the later-born interneurons have progressively more sym- while in the white matter and transplanted heterochronically into metrical terminal fields (e.g., stellate cells—Sultan and Bower, a recipient cerebellum invariably give rise to GABA interneurons, 1998). but their choice to adopt a CN, granular layer, or molecu- lar layer interneuron fate remains entirely dependent upon the GOLGI CELLS host-specific, extrinsic environment (Leto et al., 2009). Golgi cells are large interneurons of the granular layer (Palay and Few inhibitory interneurons are present in the molecular layer Chan-Palay, 1974). Golgi cell apical dendrites ramify through the at birth. While CN interneurons, are all born between E12 (Florio molecular layer and are contacted primarily by the axons of gran- et al., 2012) and P3, Golgi cells that populate the GL continue to ule cells (e.g., Geurts et al., 2003). Their axonal terminals contact divide until P4 and basket/stellate interneuron progenitors keep granule cell-mossy fiber glomeruli. Five distinct classes of Golgi proliferating through the second postnatal week [reviewed in cell have been identified based on morphology and differential Zhang and Goldman (1996); Carletti and Rossi (2008)], accord- expression patterns (various combinations of glycinergic, gabaer- ing to an inside-out progression (Leto et al., 2006). In rat, the gic, mGluR2+/−, and neurogranin+/−: Simat et al., 2007). interneurons of the inner molecular layer (= basket cells) are The origin of Golgi cells is controversial. On the one hand, born between P2 and P17 with a peak at P6, while those in the Popoff (1896)andAthias (1897) suggested the EGL was the ori- outer molecular layer (= stellate cells) are born between P4 and gin of these large neurons. This interpretation was supported P19 (peak at P10: Altman, 1972). The morphological evidence by the more recent studies of Hausmann et al. (1985), who of restriction of basket/stellate cell neurites follows a similar con- used cerebellar transplantation to provide experimental evi- tinuum. In particular for the cells located deep in the molecular dence that Golgi cells originate from the EGL. More recently,

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FIGURE 5 | Cartoon to show how basket/stellate cell parasagittal orientations might arise. Newly migrated basket/stellate cells enter the molecular layer and synapse on a small group of PCs. As the PCs extend parasagittally to form adult stripes, the basket/stellate cell arbors extend with them. As a result the shape of the final axonal arbor depends on the time they arrive: the earlier they enter the molecular layer, the more extended the arbor (= basket cell), the later they enter, the less it is extended (= stellate cells).

Chung et al. (2011) also identified a small, unique population of ZAC1-immunopositive Golgi cells, restricted to the poste- rior zone of the cerebellum that appears to derive from the EGL between E13 and E16. On the other hand, Ramon y Cajal (1911)andAltman and Bayer (1977) both concluded that Golgi cells derive from the ventricular neuroepithelium. Zhang and FIGURE 6 | Five examples (A–E) of double immunofluorescence for a Goldman (1996) reached the same conclusion based on retroviral GlyT2-EGFP transgene (Golgi cell dendrites: Zeilhofer et al., 2005)and β lineage tracing data. By this view Golgi cells, as all other GABA anti-PLC 4 (PC stripes: Sarna et al., 2006) in the adult mouse + cerebellum reveals that Golgi cell dendrites are restricted at Purkinje interneurons of the cerebellum, originate from Ascl1 progen- cell stripe boundaries. Two examples of GlyT2-EGFP+ Golgi cell dendrites itors that delaminate from the VZ into the prospective white (green: arrowheads) in the vicinity of a PLCβ4+/− stripe boundary (red): matter, exit the cell cycle, and activate Pax2. It seems probable that in 68 cases examined, the dendrite never crossed between stripes. both explanations are correct, and that two distinct populations Abbreviations: ml, molecular layer; pcl, Purkinje cell layer; gl, granular layer. Scale bar = 50 μm[FromSillitoe et al. (2008)]. of Golgi cells are present in the adult cerebellum. With the exception of the restriction to the PZ of the ZAC1+ population (Chung et al., 2011) nothing is known of the local- ization of Golgi cell subtypes to particular transverse zones or in the molecular layer. By using different markers of Golgi cell lobules. However, a different sort of patterning does occur: the dendrites and a selection of stripe antigens, the apical dendritic apical dendrites of Golgi cells show restriction at parasagittal arbors of Golgi cells were studied in the vicinity of PC stripe PC stripe boundaries (Sillitoe et al., 2008: Figure 6). Golgi cell boundaries. The conclusion was clear—fewer than 3% of Golgi apical dendrites contact the parallel fiber axons of granule cells cell dendrites cross a PC stripe boundary (Sillitoe et al., 2008).

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The mechanisms that restrict Golgi cell dendritic arbors are cerebellar interneurons”). As a result, different EGL lineages speculative. They might be prevented from crossing stripe bound- become aligned with boundaries between different PC transverse aries by structural barriers (such as those reported in the zones (Ozol and Hawkes, 1997; Hawkes et al., 1999,etc.).The somatosensory cortex—Faissner and Steindler, 1995). However, lineage boundaries are also expression boundaries, but it is not no such barriers to neurite extension are known and other clear whether these are also lineage restricted or if they are sec- axons (e.g., parallel fibers) cross parasagittal stripes unhindered. ondary responses to local PC cues. Developing Golgi cells and Alternatively, Golgi cell dendritic arbors may be restricted, via most UBCs access the PC clusters via the white matter tracts (Leto adhesion molecules or attractive/repulsive extracellular cues, as et al., 2006, 2009) and associate with specific PC clusters (e.g., they develop in concert with the PC dendrites (e.g., Hekmat Chung et al., 2009b). Therefore, as the PC clusters disperse into et al., 1989; Nagata and Nakatsuji, 1991). In this model, the newly stripes the Golgi cells and UBCs move with them, just as for born Golgi cells migrate via the white matter into the embry- mossy fiber afferent terminal fields, and therefore also become onic PC clusters (Zhang and Goldman, 1996)wheretheycontact restricted to specific stripes and zones. Subsequently, they mimic the nascent PC dendrites. Subsequently, as the PC clusters dis- the mossy fibers and relocate from the PCs to the granular layer perse into adult stripes, individual Golgi cell dendritic arbors as it matures. This also explains how Golgi cell apical dendrites would automatically become restricted to one side of a boundary become restricted to particular PC stripes (Sillitoe et al., 2008). Subsequently, as the granule cells mature, the Golgi cell den- The dispersal of embryonic PC clusters into stripes continues drites would displace from the PCs and synapse with local parallel roughly during the first three postnatal weeks (in mice). Although fibers. In this way they would retain their original topographical basket/stellate cells are born too late to interact with the embry- restriction. onic PC clusters they benefit from cluster dispersal to orient their axon arbors parasagittally. Once in situ, interneurons differentiate PURKINJE CELL ARCHITECTURE GENERATES INTERNEURON in response to local environmental cues (e.g., granule cell stripes RESTRICTION of nNOS—Hawkes and Turner, 1994). We have reviewed the evidence that cerebellar interneurons show Finally, it is interesting to speculate why parallel fibers appear anatomical and molecular restriction to zones and stripes. The to be the sole exception: why are parallel fibers not restricted? general hypothesis presented is that these restrictions come about One possibility is that it is important that they are not. Parallel through interactions with the PC architecture. In this light, it is fibers are several millimeters long (e.g., Brand et al., 1976)and worthwhile to recall briefly the hypothesis to explain how climb- as they run orthogonal to the PC stripes, they necessarily inter- ing and mossy fiber afferents become aligned with PC stripes. sect many stripes, of many different subtypes. In a nutshell, if First, the afferent fiber growth cones make direct contacts with PC boundaries were to restrict parallel fibers there would be no specific embryonic PC clusters (e.g., Sotelo and Wassef, 1991; parallel fibers! A subtler question is whether they synapse with Grishkat and Eisenman, 1995; Chédotal et al., 1997; Sotelo and all the PC dendritic arbors that they pass through. This issue Chédotal, 2005). Subsequently, the clusters disperse into stripes has not been studied experimentally. One scenario is that paral- triggered by Reelin signaling. As a result, the PC layer extends lel fibers have a primary function to distribute MF afferent input in the rostrocaudal plane, the clusters transform into stripes widely across multiple neighboring stripes, in which case they and, because the afferent terminal fields are carried along, they might be expected to synapse promiscuously with all stripes they too form stripes, which are aligned with specific PC stripes. encounter, at least initially. How that input is used is another In the case of mossy fibers, as the granular layer matures, the matter. One possibility is that PCs synapse with every PC they mossy fibers detach from their embryonic PC targets (Mason intersect but only a subset of those synapses is active: in one study, et al., 1990) and synapse instead on local granule cell dendrites. a large fraction of parallel fiber-PC synapses were found to be Hence, although the mossy fibers no longer directly contact PCs silent (Brunel et al., 2004), so sculpting in this fashion is entirely their terminal fields remain aligned with specific PC parasagittal plausible. stripes. This hypothesis is straightforwardly adaptable to the interneu- ACKNOWLEDGMENTS rons of the cerebellar cortex. First, the developing EGL spreads These studies were supported by the Canadian Institutes of Health over the surface of the cerebellar anlage, restricted by cues from Research (Richard Hawkes), and by Ataxia UK and Fondazione the underlying PCs (section “Zone and stripe boundaries restrict Berlucchi, Italy (G. Giacomo Consalez).

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G., Grumet, M., et al. (2011). Zeilhofer, H. U., Studler, B., Arabadzisz, gene expression in the embry- Citation: Consalez GG and Hawkes F3/contactin and TAG1 play antag- D.,Schweizer,C.,Ahmadi,S.,Layh, onic cerebellum. Dev. Dyn. 237, R (2013) The compartmental restric- onistic roles in the regulation of B., et al. (2005). Glycinergic neu- 1726–1735. tion of cerebellar interneurons. Front. sonic hedgehog-induced cerebel- rons expressing enhanced green flu- Neural Circuits 6:123. doi: 10.3389/fncir. lar granule neuron progenitor orescent protein in bacterial artifi- Conflict of Interest Statement: The 2012.00123 proliferation. Development 138, cial chromosome transgenic mice. authors declare that the research Copyright © 2013 Consalez and 519–529. J. Comp. Neurol. 482, 123–141. was conducted in the absence of any Hawkes. This is an open-access article Yan, X. X., Yen, L. S., and Garey, L. J. Zhang, L., and Goldman, J. E. (1996). commercial or financial relationships distributed under the terms of the (1993). Parasagittal patches in the Generation of cerebellar interneu- that could be construed as a potential Creative Commons Attribution License, granular layer of the developing rons from dividing progenitors in conflict of interest. which permits use, distribution and and adult rat cerebellum as demon- white matter. Neuron 16, 47–54. reproduction in other forums, provided strated by NADPH-diaphorase Zordan,P.,Croci,L.,Hawkes,R., Received: 21 September 2012; accepted: the original authors and source are cred- histochemistry. Neuroreport 4, and Consalez, G. G. (2008). 26 December 2012; published online: 22 ited and subject to any copyright notices 1227–1230. Comparative analysis of proneural January 2013. concerning any third-party graphics etc.

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