Expression of the IP3R1 promoter-driven nls-lacZ transgene in Purkinje cell parasagittal arrays of developing mouse cerebellum

Expression of the IP3R1 Promoter-Drivennls-LacZ Transgene in Purkinje CellParasagittal Arrays of Developing MouseCerebellum

Daisuke Furutama,1 Noriyuki Morita,2,3 Riya Takano,2 Yukiko Sekine,2

Tetsushi Sadakata,2,4 Yo Shinoda,2,4 Kanehiro Hayashi,2,4 Yuriko Mishima,2,4

Katsuhiko Mikoshiba,5 Richard Hawkes,6 and Teiichi Furuichi2,4*1First Department of Internal Medicine, Osaka Medical College, Takatsuki, Japan2Laboratory for Molecular Neurogenesis RIKEN Brain Science Institute, Wako, Japan3Department of Nutrition, Mimasaka Junior College, Tsuyama, Okayama, Japan4JST-CREST, Kawaguchi, Saitama, Japan5Laboratory for Developmental Neurobiology, SORST-JST, RIKEN Brain Science Institute, Wako, Japan6Department of Cell Biology and Anatomy, and Genes and Development Research Group,Faculty of Medicine, The University of Calgary, Calgary, Alberta, Canada

The cerebellar Purkinje cell monolayer is organized intoheterogeneous Purkinje cell compartments that havedifferent molecular compositions. Here we describe atransgenic mouse line, 1NM13, that shows heterogene-ous transgene expression in parasagittal Purkinje cellarrays. The transgene consists of a nuclear localizationsignal (nls) fused to the b-galactosidase (lacZ) compositegene driven by the type 1 inositol 1,4,5-trisphosphate re-ceptor (IP3R1) gene promoter. IP3R1-nls-lacZ transgeneexpression was detected at a single Purkinje cell levelover the surface of a whole-mount X-gal-stained cerebel-lum because of nuclear accumulation of the nls-lacZactivity. Developing cerebella of 1NM13 mice showedstripe-like X-gal staining patterns of parasagittal Purkinjecell subsets. The X-gal stripe pattern was likely deter-mined by an intrinsic property as early as E15 andshowed increasing complexity with cerebellar develop-ment. The X-gal stripe pattern was reminiscent of, butnot identical to, the stripe pattern of zebrin II immunore-activity. We designated the symmetrical X-gal-positive(transgene-positive, Tg1) Purkinje cell stripes about themidline as vermal Tg11, Tg2(a, b)1 and Tg3(a, b)1 stripesand hemispheric Tg4(a, b)1, Tg5(a, b)1, Tg6(a, b, c)1,and Tg7(a, b)1 stripes, where a, b, and c indicate sub-stripes. We also assigned three parafloccular substripesTg8(a, b, c)1. The boundaries of X-gal stripes at P5 wereconsistent with raphes in the Purkinje cell layer throughwhich granule cells migrate, suggesting a possible asso-ciation of the X-gal stripes with raphe formation. Ourresults indicate that 1NM13 is a good mouse model witha reproducible and clear marker for the compartmentali-zation of Purkinje cell arrays. VVC 2010 Wiley-Liss, Inc.

Key words: cerebellar compartment: IP3 receptor; lacZtransgene; Purkinje cell zones; zebrin

The mammalian cerebellum is compartmentalizednot only in terms of gross anatomy, afferent and efferentprojections, and synaptic connectivity (Gravel et al.,1987; Sotelo and Wassef, 1991; Voogd, 1995; Voogdand Glickstein, 1998; Llinas and Walton, 1998; Appsand Garwics, 2005; Apps and Hawkes, 2009) but also interms of the molecules that are expressed in neurons(Hawkes, 1997; Hawkes and Eisenman, 1997; Herrupand Kuemerle, 1997; Oberdick et al., 1998; Armstrongand Hawkes, 2000; Armstrong et al., 2001; Sillitoe andJoyner, 2007). It is known that heterogeneous Purkinjecell subsets with different molecular compositions aredistributed into transverse zones that are further subdi-vided into parasagittal stripes (see, for example, Ozolet al., 1999; Apps and Hawkes, 2009). Many reportshave shown alternating Purkinje cell stripes expressing orlacking various molecules; among these, the antigenzebrin II (aldolase C) has been studied in great detail(Hawkes et al., 1985; Brochu et al., 1990). There is anapparent correlation among the molecular, morphologi-

Additional Supporting Information may be found in the online version

of this article.

Contract grant sponsor: Japanese Ministry of Education, Culture, Sports,

Science, and Technology (to N.M., T.F.); Contract grant sponsor: Japan

Science and Technology Agency (to T.F.); Contract grant sponsor: Japan

Society for the Promotion of Science (to T.F.); Contract grant sponsor:

Canadian Institutes of Health Research (to R.H.).

*Correspondence to: Teiichi Furuichi, PhD, Laboratory for Molecular

Neurogenesis, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako, Sai-

tama 351-0198, Japan. E-mail: [email protected]

Received 3 February 2010; Revised 8 April 2010; Accepted 26 April

2010

Published online 14 July 2010 in Wiley Online Library

(wileyonlinelibrary.com). DOI: 10.1002/jnr.22451

Journal of Neuroscience Research 88:2810–2825 (2010)

' 2010 Wiley-Liss, Inc.

cal, and physiological compartments of Purkinje cells.For example, the zebrin II-immunopositive and -immu-nonegative Purkinje cell stripes were shown to corre-spond to afferent and efferent projection patterns(Hawkes, 1997; Voogd and Glickstein, 1998; Voogdet al., 2003; Sugihara and Shinoda, 2004; Sugihara andQuy, 2007), to somatotopic maps revealed by tactile(Chockkan and Hawkes, 1994; Hallem et al., 1999) orelectrical (Chen et al., 1996) face stimulation, and tomolecular layer inhibition evoked by parallel fiber stimu-lation (Gao et al., 2006). The organization of parasagittalPurkinje cell stripes is thought to be an intrinsic, cell-au-tonomous property that is conferred when or shortly af-ter these cells arise in the subventricular zone of thefourth ventricle (Leclerc et al., 1988; Wassef et al., 1990;Seil et al., 1995). Correspondingly, particular subsets ofparasagittally clustered Purkinje cells were shown tohave the same birth date (Hashimoto and Mikoshiba,2003). Possible involvement of cell signaling molecules,including the cell–cell recognition molecules cadherin(Arndt and Redies, 1998) and Eph-ephrin (Karam et al.,2000) and the reelin receptors Apoer2 and Vldlr (Lar-ouche et al., 2008), in the formation of Purkinje cellclusters has been shown. Several transcription factorshave also been implicated in differential transcriptionalregulation between Purkinje cell stripes (Herrup andKuemerle, 1997; Oberdick et al., 1998; Chung et al.,2008; Sillitoe et al., 2008). However, the molecularmechanism underlying Purkinje cell stripe formationremains unclear.

The inositol 1,4,5-trisphosphate (IP3) receptor(IP3R) is an IP3-gated Ca21 release channel (Furuichiet al., 1994; Furuichi and Mikoshiba, 1995). In the cen-tral nervous system, type 1 IP3R (IP3R1) is the predom-inant family member and is highly expressed in Purkinjecells (Maeda et al., 1989; Furuichi et al., 1989, 1993).We previously generated transgenic mouse lines carryinga transgene composed of a nuclear localization signal(nls)-fused Escherichia coli b-galactosidase (b-gal) gene(lacZ) whose expression is controlled by the mouseIP3R1 gene promoter (Furutama et al., 1996). In thepresent report, we describe that one of the transgeniclines, 1NM13, displays Purkinje cell heterogeneity inIP3R1-nls-lacZ transgene expression during cerebellar de-velopment. X-gal (a chromogenic substrate for b-gal)staining patterns of 1NM13 mouse cerebellumresembled, but were slightly different from, parasagittalPurkinje cell stripe patterns of the lacZ transgene drivenby the Purkinje cell-specific protein L7/pcp2 (Oberdicket al., 1991, 1993; Symeyne et al., 1991; Vandaele et al.,1991) and olfactory marker protein (OMP; Nunzi et al.,1999) gene promoters. We characterized the compart-mentalization of Purkinje cell stripes in terms of IP3R1-nls-lacZ transgene expression during cerebellar develop-ment. Our results showed that 1NM13 mice are a goodmouse model with a reproducible and clear, standardmarker for studying the compartmentalization of Pur-kinje cell arrays in the Purkinje cell monolayer of thecerebellum.

MATERIALS AND METHODS

Animals

All experimental protocols were approved by theRIKEN Institutional Animal Care and Use Committee. Con-struction of the IP3R1 promoter nls-lacZ transgenic mice hasbeen described previously (Furutama et al., 1996). The trans-gene encodes an E. coli lacZ protein fused to the nuclear local-ization signal (nls) of the rat glucocorticoid receptor (Picardand Yamamoto, 1987) and is regulated by the IP3R1 genepromoter. These lines, TgN(IP3R1nls-lacZ), were maintainedon a B6C3F1 genetic background by mating hemizygoticmales with wild-type females (B6C3F1; Nippon SLC, Shi-zuoka, Japan). Transgenic mice were identified by analyzingDNA extracted from tail biopsies, using either polymerasechain reaction (PCR) or Southern blot hybridization analysiswith a lacZ DNA probe 3-kb BamHI fragment as describedpreviously (Furutama et al., 1996). For all experiments, hemi-zygotes of the transgenic mouse line B6C3F1-TgN(IP3R1nls-lacZ)1NM13Furu, abbreviated as 1NM13, were used to avoidany possible phenotypes resulting from nonhomologous inser-tion of the transgene into the chromosome.

Detection of b-Galactosidase Activity

IP3R1-nls-lacZ transgene expression was detected by usingan X-gal (5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside)staining protocol, basically as described by Allen et al. (1988)and Oberdick et al. (1994). In brief, transgenic mice were anes-thetized with ether and then perfused transcardially sequentiallywith phosphate-buffered saline (PBS), 4% paraformaldehyde,and 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). Cer-ebella or brains were dissected and postfixed with the same fixa-tive for 1 hr on ice. After rinsing in PBS three times for 10 mineach, tissues were incubated in X-gal solution [0.5 mg/ml X-gal(Takara Shuzo, Kyoto, Japan), 3 mM potassium ferricyanide, 3mM potassium ferrocyanide, 1 mM MgCl2 in 0.1 M phosphatebuffer, pH 7.0] at 378C for 30 min to several hours or at roomtemperature for 12–24 hr and then rinsed in PBS several times.Histochemical detection of b-gal activity in cryostat sections(coronal or parasagittal) of transgenic mouse cerebella was per-formed as described previously (Furutama et al., 1996).

Immunohistochemistry

After the b-gal histochemical procedure describedabove, sections were further analyzed for detection of immu-noreactivity for IP3R1 or zebrin II. In brief, after three washeswith PBS, X-gal-stained sections were processed by the fol-lowing immunostaining steps, each of which, except for step2, was followed by three washes with PBS for 5 min each: 1)0.3% H2O2 in methanol for 30 min, 2) 2% normal goat serum(Vector Laboratories, Burlingame, CA) and 2% normal horseserum (Vector) in PBS for 1 hr, 3) anti-IP3R1 rat monoclonalantibody (mAb4C11; Maeda et al., 1989) or zebrin II mousemonoclonal antibody (Brochu et al., 1990; 10-fold dilution ofthe hybridoma supernatants) for 1 hr, 4) biotinylated goatanti-rat IgG (for IP3R1 detection) or anti-mouse IgG (forzebrin II detection; Vector) for 30 min, 5) horseradish peroxi-dase-conjugated avidin (Vector) for 30 min, and 6) 0.02%H2O2 and 0.1% 3,30-diaminobenzidine in PBS. Whole-mount

IP3R-lacZ in Purkinje Cell Stripes 2811

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zebrin II immunocytochemistry was performed as describedby Sillitoe and Hawkes (2002).

Organotypic Cultures of Whole Cerebella

Organotypic cultures of whole cerebella were preparedfrom 1NM13 mice at various developmental stages and main-tained in culture medium (Stoppini et al., 1991) consisting of 50%MEM, 25% horse serum, and 25% Hank’s solution buffered topH 7.2 by addition of 5 mMTris and 4 mMNaHCO3, with pen-icillin and streptomycin added. Cultures were kept in an incuba-tor with 5% CO2/95% air at 378C. When necessary, cultureswere treated with 30 mMKCl, 1 lM tetrodotoxin (TTX; Sigma,St. Louis, MO), or 10 lM bicuculline (BCC; Sigma). In somecultures, whole cerebella were placed on granule cell feeder layersthat were prepared from postnatal day (P) 5 cerebella of ICRmice(Nippon SLC) as described previously (Yuzaki et al., 1996).

RESULTS

Parasagittal Stripe Expression of the IP3R1-nls-lacZTransgene as Revealed by Whole-MountX-Gal Staining

We previously established seven transgenic mouselines carrying an nls-lacZ composite gene downstream ofthe 50-flanking promoter region of the mouse IP3R1 gene(from nucleotide positions –528 to 1169; Fig. 1A; Furu-tama et al., 1996). Southern blotting analysis showed dif-

ferences in chromosomal sites and copy numbers of theIP3R1-nls-lacZ transgenes among these mouse lines (Fig.1B). Thus, we expected to find differences in transgeneexpression across the brain regions among these mouselines; such differences are sometimes reported with trans-genic approaches (Bonnerot et al., 1990). We thereforeinvestigated expression of the transgene in developingmouse brains by whole-mount staining with X-gal(Oberdick et al., 1994). One of these seven lines, desig-nated 1NM13, showed a unique heterogeneous pattern ofX-gal staining over the surface of the cerebellum, in con-trast to the almost uniform staining patterns found in theother lines (data not shown), suggesting a differentialtransgenic effect. The tentatively assigned numbers of1NM13 whole-mount X-gal-positive (that is, transgene-positive) stripes are summarized in Figure 2 in comparisonwith the whole-mount zebrin II immunostaining stripes.This distribution has been reproducible among individualsand across generations of 1NM13 mice for more than 13years since generation of the line.

Stripe Expression Patterns of the IP3R1-nls-lacZTransgene in 1NM13 Mouse Cerebella Resemble,but Are Not Identical to, Those of Zebrin II

Previous zebrin II immunostaining studies of thevermis have revealed four transverse expression domains

Fig. 1. IP3R1 promoter-driven nls-lacZ transgene and chromosomalinsertions in transgenic mouse lines. A: Expression of the nls-lacZtransgene controlled by the 50-flanking region from nucleotide posi-tions –528 to 1169 of the IP3R1 gene including the promotersequence (–528 to –1), the initiation site of the transcription (11), anda portion of the first exon (11 to 1169). Within the promoter region,there are many consensus sites for transcriptional regulation (Furuichiet al., 1989; Furutama et al., 1996), and in vitro transcription assayshave demonstrated that an E-box binds to a basic helix-loop-helix,

NeuroD-related factor (NDRF; Konishi et al., 1997, 1999). EV, cut-ting sites of EcoRV. B: Variations in chromosomal locations andtransgene copy numbers in seven transgenic lines were analyzed bySouthern blot hybridization (EcoRV-digested tail-biopsy DNAs) usingthe lacZ gene as a probe. The numbers along the left side show themolecular standard sizes in kb, and those along the right side show thesizes of two bands (4.4 and 1.8 kb) expected as derivatives from thetransgene. Asterisks indicate DNA fragments probably derived from agap between the transgene and the inserted chromosome.

2812 Furutama et al.


Fig. 2. Schematic representation of the Purkinje cell stripe patternsrevealed by using 1NM13 whole-mount X-gal staining. Whole-mount X-gal-positive (transgene-positive) stripes are referred to asTg1 stripes and are tentatively assigned the numbers 1–7 symmetri-cally with respect to the midline (for example, Tg11, Tg2a1,Tg2b1, where a, b, and c indicate substripes). Paraflocular stripes arealso designated as Tg81 stripes, consisting of three substripes. A:Dorsal views of antizebrin II staining patterns in the adult mouse cer-ebellum (the paraflocculus was removed) and X-gal staining patternsin the P14 1NM13 cerebellum are shown on the left and right,respectively. LS, lobulus simplex; CI, crus I of ansiform lobule; CII,crus II of ansiform lobule; PL, paramedian lobule; CP, copula pyra-midis. Lobules V, VIa, VIb, VII, VIII, IX, and X are indicated. Tra-

verse zones AZ (anterior zone), lobules I–V; CZ (central zone),lobules VI–VII; PZ (posterior zone), lobules VIII–anterior IX; NZ(nodular zone), lobules posterior IX–X (Ozol et al., 1999). B: Sche-matic representations of anti-zebrin II staining patterns and 1NM13X-gal staining patterns shown in A. Major X-gal-positive stripes aresimilar but not identical to zebrin II-immunopositive stripes (Ozolet al., 1999; Sillitoe and Hawkes, 2002). However, the 1NM13X-gal-positive (transgene-positive) stripes Tg11, Tg2(a, b)1, Tg3(a,b)1, Tg4(a, b)1, Tg5(a, b)1, Tg6(a, b, c)1, Tg7(a, b)1, and Tg8(a,b, c)1 are slightly more complicated than the zebrin II-immunoposi-tive stripes (P11, P21, P31, P4a1, P4b1, P5a1, P5b1, P61, andP71).



or zones: the anterior zone (AZ: lobules I–V), the cen-tral zone (CZ: lobules VI, VII), the posterior zone (PZ:lobules VIII, anterior IX), and the nodular zone (NZ:posterior IX, X; Ozol et al., 1999; Armstrong et al.,2000; Sillitoe and Hawkes, 2002; Apps and Hawkes,

2009; Fig. 2). Each zone can be further subdivided intothree parasagittal zebrin II-positive (P1) stripes (P11,P21, and P31) symmetrical about the midline (Fig. 2).1NM13 cerebella at P5 (Fig. 3A–D) and P14 (Figs. 3E–H, 4A–F) also showed similar symmetric zonation carry-

Fig. 3. Whole-mount X-gal staining patterns of 1NM13 cerebella at P5 and P14. Whole brains ofP5 and P14 1NM13 transgenic mice were stained with X-gal to reveal IP3R1-nls-lacZ transgeneexpression. After X-gal staining, cerebella were detached from the rest of the brain and photo-graphed from rostral (A,E), dorsal (B,F), caudal (C,G), and ventral (D,H) angles. Black and whitearrows indicate the boundaries of stripes and substripes, respectively. Scale bars 5 1 mm.



ing three major whole-mount X-gal positive (transgene-positive Tg1) stripes, designated as Tg11, Tg21, andTg31 (Fig. 2). Four major hemispheric X-gal-positivestripes, designated as Tg41, Tg51, Tg61, and Tg71, in

the crus II of ansiform lobule (CII) and paramedianlobule (PL) of 1NM13 cerebella (Fig. 4H,G) alsoresembled hemispheric zebrin II stripes P41, P51, P61,and P71 (Fig. 2; Sillitoe and Hawkes, 2002).

Fig. 4. Higher magnification of vermal and hemispheric X-gal staining patterns observed in 1NM13cerebella at P14. A–E: Dorsal views of vermal lobules. F: Ventral view of nodular lobules. G,H:Dorsal views of hemispheres. Reproducible expression stripes are numbered. Dotted lines in A–Eindicate the position of the midline and the paravermian stripes. Arrows and vertical lines indicatethe boundaries between X-gal-positive stripes and substripes, respectively. Scale bars 5 0.5 mm.



The 1NM13 X-gal stripe patterns in vermalregions, however, are not identical to the stripe patternsdefined by zebrin II expression. In lobule V of the AZ,zebrin II immunostaining showed a narrow stripe (P11)present symmetrically, with one astride the midline andat least two stripes (P21 and P31) laterally to either side(Fig. 2B), whereas 1NM13 X-gal staining was uniquelyshaped into five clusters: Tg11, Tg2(a, b)1, and Tg3(a,b)1, where a and b indicate substripes (Fig. 4A–F, Supp.Info. Fig. 1). In the CZ, zebrin II immunostainingshowed three stripes, P11, P21, and P31, in lobule VIaand a single broad band in lobules VIb and VII (Fig.2B). 1NM13 X-gal staining labeled more stripes,namely, Tg11, Tg2(a, b)1, and Tg3(a, b)1 in lobule VIa(Fig. 4B, Supp. Info. Fig. 1), and showed widespreadand loosely clustered (ambiguous boundary) stripesTg11, Tg21, and Tg31 in lobule VIb (Fig. 4C, Supp.Info. Fig. 1) and stripes Tg11, Tg21, and Tg31 (orTg21 and Tg31 probably because of a missing stripeTg11) in lobule VII (Fig. 4D, Supp. Info. Fig. 1). In thePZ, zebrin II immunostaining again became striped,with a midline stripe P11, stripes P21 and P31 morelaterally at each side, and a stripe P41 in the paravermianregion (Fig. 2A,B). 1NM13 X-gal staining exhibitedsimilar stripes, Tg11 to Tg4a1, in lobule VIII (Fig. 4D)but indicated more stripes, namely, Tg11, Tg2(a, b)1,Tg3(a, b)1, and Tg41, in lobule IX (anterior IX; Fig.4E). In the NZ, zebrin II also had four major stripes,P11 to P41 (Fig. 2), and 1NM13 X-gal staining showedstripes Tg11, Tg2(a, b)1, Tg3(a, b)1, and Tg41 (poste-rior IX; Fig. 4F). In hemispheric regions, 1NM13 X-galstaining showed more complex stripe patterns, specifi-cally, stripes Tg4(a, b)1, Tg5(a, b)1, Tg6(a, b, c)1, andTg7(a, b)1 in the lobules CII and PL (Fig. 4G,H, Supp.Info. Fig. 2), in comparison with the patterns of zebrinII immunostaining, namely, stripes P4a1, P4b1, P5a1,P5b1, P61, and P71 (Fig. 2). X-gal stripes in the otherhemispheric lobules, lobulus simplex (LS), crus I of ansi-form lobule (CI), and copula pyramidis (CP), exhibitedpatterns similar to those of the CII and LP, although theCI had high density and broad positive cell stripes sepa-rated by very narrow and irregular boundaries (Fig. 4G,Supp. Info. Fig. 2). Taken together, these results indicatethat the IP3R1-nls-lacZ transgene in 1NM13 mouse cer-ebella is expressed in subsets of Purkinje cells clusteredin the symmetrical parasagittal X-gal-positive stripesTg11, Tg2(a, b)1, Tg3(a, b)1, Tg4(a, b)1, Tg5(a, b)1,Tg6(a, b, c)1, and Tg7(a, b)1 about the midline [theparaflocculus has substripes Tg8(a, b, c)1 as described inthe next section].

We next analyzed the transgene expression insideof the 1NM13 mouse cerebella by histochemical X-galstaining of coronal sections (Fig. 5). The X-gal stainingpatterns at P13 showed that some stripes are composedof intermingled heterogeneous Purkinje cells, which areX-gal-positive (solid arrows) and -negative (open arrows)cells (tg1 region in Fig. 5A) and that others includeweakly positive cells (left tg1 region in Fig. 5B) orstrongly positive cells (right tg1 region in Fig. 5B).

Notably, double staining patterns for X-gal and zebrin IIat P12 showed little coherence between X-gal-positive(tg1) and zebrin II-immunopositive (zb1) Purkinje cells(Fig. 5C–E), although the major whole-mount stripepatterns seemed to be similar between X-gal and zebrinII (Fig. 2). In lobule III, zebrin II and X-gal double-pos-itive cells (zb1/tg1) and zebrin II single-positive cells(zb1/tg2) were intermingled within the zebrin II P21

stripe. An X-gal single-positive cluster was also observedbetween the zebrin II P21 and P31 stripes. In lobulesIV and V, no tg1 cells were observed at a position cor-responding to the P21 stripe, although an X-gal-positivestripe was present adjacent to the P21 stripe (Fig. 5E).Taken together, these results suggested more compli-cated heterogeneities of individual Purkinje cells at mo-lecular levels in arrays of the PCL. However, further sys-tematic study is necessary to determine the Purkinje cellheterogeneities represented by these two stripe markers,IP3R1-nls-lacZ and zebrin II.

Developmental Changes in Purkinje Cell StripePatterns Expressing the IP3R1-nls-lacZ Transgene

Developmental changes in the IP3R1-nls-lacZ trans-gene expression in 1NM13 cerebella were analyzedusing whole-mount specimens (E15, P0, P3, P5, P12,P13 and P14; Fig. 6). Nuclear X-gal staining of 1NM13cerebella clearly revealed distinct expression domainsassociated with specific Purkinje cell clusters as early asE15 (Fig. 6A) and showed an increasing complexity ofPurkinje cell stripe patterns during cerebellar develop-ment. Coronal cerebellar sections at E17 also showed aconsistent pattern consisting of symmetric arrays of Pur-kinje cell nuclei stained with X-gal (Fig. 6B). More Pur-kinje cells became X-gal-positive until they coveredalmost the whole cerebellar cortex at the end of the sec-ond postnatal week, thereby rendering the stripe boun-daries unclear after P14. Thus, adult 1NM13 mice didnot show clear stripe patterns by whole-mount X-galstaining, as reported previously (Furutama et al., 1996).This is a marked difference from the L7bG3 strain ofL7/pcp2-lacZ transgenic mice, which shows a stripedexpression pattern even on P140 (Oberdick et al., 1993;Ozol et al., 1999).

Vermes. Two major symmetrical stripes wereobserved in the developing vermes from E15 to P14(indicated by asterisks in Fig. 6A–G). The maturation ofthe striped expression pattern in the 1NM13 cerebellarvermis could be followed in each transverse zone. AZ:Three major Tg1 stripes (Tg11, Tg21, and Tg31) wereseen in the anterior lobe vermis by P5 (Fig. 3A). Inlobule V, clusters developed into an adult pattern at P12(Fig. 6F) with unique four substripes Tg2(a, b)1, andTg3(a, b)1 at P14 (Figs. 3E, 6G). CZ: In presumptivesublobule VIa, clusters became evident at P5 (Figs. 2B,6E). A characteristic stripe pattern Tg2(a, b)1 and Tg3(a,b)1 appeared in lobule VIa at P14 (Figs. 4B, 6G). Inpresumptive lobules VIb and VII, widespread X-gal-pos-itive clusters became obvious by P12 (Fig. 6F), but



lobules VIb–VII appeared faintly striped at P14 (Figs.3B,C, 6G). PZ: In lobule VIII, there was a weakly la-beled midline stripe Tg11 by P3 (Fig. 6D) and two ver-mal stripes laterally to each side, a strongly labeled stripeTg21 and a weakly labeled stripe Tg31 (Fig. 6A–G),flanked by the strongly labeled paravermian stripe

Tg4a1. Stripes Tg21 and Tg41 (open asterisk and slolidasterisk in Fig. 6, respectively) were already clear as earlyas E15 (Fig. 6A). In lobule IX, two prominent stripes(presumptive stripes Tg21 and Tg4a1) appeared at E15(indicated by asterisks in Fig. 6A), became much broaderat P3–P5 (Fig. 6D,E), and almost fused with each other

Fig. 5. Comparison of X-gal staining and zebrin II immunostainingpatterns in 1NM13 cerebellar sections. A,B: Coronal sections of1NM13 cerebella at P13 were stained with X-gal. Purkinje cell layershave heterogeneous arrays including X-gal-positive (tg1) and -nega-tive (tg2) stripes. There is heterogeneity in the tg1 stripes: some tg1

stripes are composed of heterogeneous Purkinje cells, which are X-gal-positive (solid arrows) and -negative (open arrows) cells (tg1 inA), and others are composed of weakly positive cells (left tg1 in B)

or strongly positive cells (right tg1 in B). C–E: Coronal sections of1NM13 cerebella at P12 were double stained with X-gal (blue) andantizebrin II antibody (peroxidase: brown). Regions outlined by rec-tangles in C are shown at higher magnification in D,E. Well-definedzebrin II-immunopositive (P11, P21 and P31) and -negative (P12

and P22) stripes are shown. Zb1, zebrin II-immunopositive cells;tg1, X-gal-positive; tg2, X-gal-negative. Scale bars 5 25 lm in B(applies to A,B); 200 lm in C; 50 lm in E (applies to D,E).



Fig. 6. Developmental changes in X-gal staining patterns of 1NM13cerebella. A–G: Representative whole-mount X-gal staining patternsof 1NM13 cerebella at E15, P0, P3, P5, P12, and P14 are shown. Arepresentative X-gal staining pattern of 1NM13 cerebellar coronal sec-tion at E17 is also shown. Characteristic stripes are indicated by solidand open asterisks (vermal stripe Tg21 and paramedian stripe Tg4a1,respectively) and solid and open arrowheads (hemispheric stripesTg5a1 and Tg61, respectively). Hemispheric lobules (LS, lobulus sim-plex; CI, crus I of ansiform lobule; CII, crus II of ansiform lobule; PL,

paramedian lobule; CP, copula pyramidis), paraflocculus (PF), and ver-mal lobules (V, VI, VII, VIII, and IX) are indicated. Arrows show asmall furrow indicative of the beginning of sublobulation in lobules CIand CII at P14. H–N: Representative whole-mount X-gal stainingpatterns of the paraflocculus region in 1NM13 mice at P0, P3, P5,P13 and P14 are shown. In the paraflocculus, there are three substripesTg8(a, b, c)1 divided by the strongly labeled rostromedial stripeTg8b1 indicated by arrows. PF, paraflocculus; FL, flocculus. Scale bars5 0.5 mm in A–C,H–N; 1 mm in D–G.



at P12 (Fig. 6F). However, lobule IX appeared to havethree subbands at P5 (Figs. 3C, 6E). Four wide andhigh-density Tg1 substripes Tg2(a, b)1 and Tg3(a, b)1

were still distinguishable by boundaries of X-gal-negativenarrow bands at P14 (Figs. 3G, 4E,F, 6G). NZ: Theposteroventral view of lobules IX and X showed sixstripes Tg11, Tg2(a, b)1, Tg3(a, b)1, and Tg41 at P5(Fig. 3D) and at least six stripes Tg11, Tg2 (a, b)1,Tg3(a, b)1, and Tg41 at P14 (Fig. 3H, 4E,F). The Tg1

stripes assigned the same number are considered to origi-nate from the same Purkinje cell clusters but are notexactly matched to one another among transverse zonesand lobules in terms of their shapes, such as stripe widthand positive Purkinje cell density within stripes. Thestripe patterns between the lobules CII and LP areexceptionally similar (Fig. 4H, Supp. Info. Fig. 2).

Hemispheres. In the hemispheres, distinct Pur-kinje cell stripes were already conspicuous as early asE15 and became more obvious between P0 (Fig. 6C)and P3 (Fig. 6D). A paravermian X-gal-positive stripewith high intensity (indicated by an open asterisk in Fig.6) developed into stripe Tg4a1 (Fig. 6C–G). Therewere two hemispheric stripes with high and medium X-gal staining intensities (indicated by open arrowhead andfilled arrowhead in Fig. 6, respectively). The formerstripe developed into stripe Tg61 (Fig. 6C–G) and couldbe subdivided into three substripes Tg6(a, b, c)1 at P14(Fig. 4G,H, Supp. Info. Fig. 2), whereas the latter devel-oped into stripe Tg5a1 (Figs. 3G–I, 6C–G, Supp. Info.Fig. 2). Although the early fundamental staining patternsbecame complicated with the lobulation and expansionof the hemispheres, the major stripes were sustained untilthe second postnatal week.

Paraflocculi. A basic stripe pattern was evidentin the presumptive paraflocculus of the posterior lobe asearly as the day of birth (Fig. 6H) and was largelyunchanged by subsequent dynamic morphogeneticevents (Fig. 6H–L). On P0 (Fig. 6H), in the posterolat-eral region of immature lobule IX, there was a sharptranslobular cluster of high expression (indicated by thearrow in Fig. 6H–N) flanked by dispersed mid-intensityclusters. This whole region moved laterally on P3 (Fig.6I) and protruded with a slight anterohorizontal twistoutside the cerebellum on P5 (Fig. 6J), leading to theformation of a parafloccular bulge. The paraflocculusswelled further, increasing its volume, and extendedposterolaterally around the second postnatal week. As aresult, three distinct X-gal staining domains, parafloccu-lar stripes, became apparent: anterodorsal stripe Tg8a1

and posteroventral stripe Tg8c1 (with medium and lowstaining intensity, respectively), separated by a high-in-tensity stripe Tg8b1 (Fig. 6M,N). The stripes were ori-ented anterior-posterior in the early stages but shifted ina mediolateral direction in older animals. The paraflocc-ular stripe pattern of 1NM13 transgene expression differsin both its timing and arrangement from that describedfor OMP-lacZ mice (two zones, an X-gal-positive dorsalzone and an X-gal-negative ventral zone, develop atP10–P20; Nunzi et al., 1999).

Purkinje Cell Clustering With Sharp BoundariesBetween Transgene-Positive and -NegativeCell Arrays

To compare the expression patterns of the IP3R1-nls-lacZ transgene and endogenous IP3R1, coronal sec-tions of 1NM13 mouse cerebella at P5 were doublelabeled with X-gal and anti-IP3R1 antibody (Fig. 7).IP3R1 immunoreactivity was specifically localized in thesomata and growing dendrites of all Purkinje cells,whereas the X-gal reaction product was observed in asubset of Purkinje cell nuclei (Fig. 7A,B), again suggest-ing that the heterogeneity of IP3R1-nls-lacZ transgeneexpression regulation among Purkinje cell subpopula-tions likely is due to a transgenic effect. It is notable thatthe boundaries between X-gal-positive and -negativePurkinje cell arrays were very sharp, suggestingextremely tight on/off regulation of transgene expressionat the boundaries.

At P5, the Purkinje cell layer (PCL) had gaps thatwere nonreactive to both types of staining (shown byarrows in Fig. 7). Interestingly, some of these unstainedgaps were consistent with boundaries between X-gal-positive and -negative Purkinje cell arrays (Fig. 7A).These gaps were occupied by a stream of small cellsfrom the molecular layer (ML) to the internal granularlayer (IGL) rather than by Purkinje cell somata (Fig.7C), suggesting that they probably correspond to theraphes through which some granule cells migrate fromthe external granular layer (EGL) to the IGL (Arndtet al., 1998; Karam et al., 2000, 2001; Luckner et al.,2001; Redies et al., 2002). Distinct raphes were notobserved at later stages. These results suggest that theheterogeneous regulation of transgene expression is pos-sibly associated with raphe formation in early develop-mental stages. Similar gaps were also present withinIP3R1 single-positive (X-gal-negative) arrays (Fig. 7B),suggesting the presence of more distinct Purkinje cellarrays having different molecular organizations in thePCL.

Parasagittal Stripe Patterns of TransgeneExpression in Organotypic Cerebellar Cultures

Previous studies have reported that the stripeexpression patterns of zebrin (Leclerc et al., 1988; Wassefet al., 1990; Seil et al., 1995) and the L7/pcp2-lacZ trans-gene (Oberdick et al., 1993) are intrinsic, cell-autono-mous properties of Purkinje cells, which are conferred atthe birth of Purkinje cells or shortly after it. Our resultssuggested that Purkinje cells express the IP3R1-nls-lacZtransgene by E15, well after their final cell division hasbeen completed (E10–E13: Uzman, 1960; Miale andSidman, 1961; Inouye and Murakami, 1980). To investi-gate whether the 1NM13 transgene expression is cell au-tonomous and is unchanged by environmental condi-tions, we analyzed the X-gal staining patterns of 1NM13mouse cerebella in in vitro culture systems. 1NM13 cer-ebella dissected at E15 (Fig. 8A), E17 (Fig. 8B), and P3(Fig. 8C) were assessed by whole-mount X-gal staining



after organotypic culturing for different days in vitro(DIV). The representative X-gal stripe patterns weremaintained despite in vitro culturing without extracere-bellar afferents, although the overall structure of the cul-tured cerebella became distorted because of the out-growth of cells, and this was probably accompanied bycell death. X-gal stripe patterns seemed to be a littlemore pronounced in their numbers and staining inten-sities with time in culture, as in the case of L7/pcp2-lacZ(Oberdick et al., 1993). E15 cerebella, cultured togetherwith brainstems from which some afferent inputs mightbe derived with time in culture (Paradies et al., 1996),also showed no significant difference from the in vivoexpression pattern after 3 DIV (data not shown). Sec-tions prepared from these whole-cerebella cultures weresubjected to immunohistochemical staining for IP3R1:Purkinje cell dendrites, axons, and somata were allstrongly immunoreactive, suggesting good preservationof Purkinje cells inside the cerebella cultured withinthese time frames (data not shown).

We next analyzed a possible role for membraneexcitability, based on the fact that the complexity of thezonal pattern increases during postnatal developmentalstages during which the synaptic connectivity of cerebel-

lar circuitry is actively formed. P0 cerebella of 1NM13mice were cultured for 3, 5, and 10 DIV in the presenceof 30 mM KCl (membrane depolarization), 1 lM tetro-dotoxin (TTX, a sodium channel blocker), or 10 lMbicuculline (BCC; a GABAA receptor inhibitor). Cere-bella treated with high KCl expressed the typical in vivoX-gal staining pattern, with an apparently higher inten-sity of staining but no significant change in the pattern(Fig. 9A). Treatments with TTX (Fig. 9B) and BCC(Fig. 9C) did not change the basic pattern, althoughboth caused a gross disturbance of cerebellar structure.

In dissociated cultures of newborn cerebellum,excess granule cells likely have a trophic effect on Pur-kinje cell survival (Yuzaki et al., 1996). To test whetherexogenous granule cells modify the transgene expressionpattern, we analyzed P0 1NM13 mouse cerebella afterculturing for 3, 5, and 10 DIV on a granule cell feederlayer and found no significant change in X-gal patterns(Fig. 9D).

DISCUSSION

The 1NM13 transgenic mouse line carrying theIP3R1-nls-lacZ transgene allows us to visualize the para-

Fig. 7. Double staining of P5 1NM13 cerebellar sections by X-galreaction and IP3R1 immunoreaction. A–C: Horizontal sectionsthrough the 1NM13 cerebella at P5 were double stained with X-gal(blue) and anti-IP3R1 monoclonal antibody 4C11 (brown). EGL,external germinal layer; ML, molecular layer; PCL, Purkinje celllayer; IGL, internal granular layer; WM, white matter. Arrows indi-

cate gaps in the PCL that were not stained with X-gal or anti-IP3R1. Arrow in C represents a putative migrating granule cellstream between the X-gal-positive and -negative clusters, whichprobably corresponds to a granule cell raphe, as previously reported(Luckner et al., 2001; Karam et al., 2001). Scale bars 5 100 lm inA; 200 lm in B; 50 lm in C.



Fig. 8. X-gal stripe patterns in organotypic cultures of 1NM13 cerebella. Cerebella were dissectedfrom E15 (A), E17 (C), and P3 (D) 1NM13 mice and were cultured for 0, 6, and 8 DIV (A); 4,6, and 11 DIV (B); or 0, 2, and 7 DIV(C), respectively. After organotypic culturing, cerebellawere stained with X-gal. Scale bars 5 0.5 mm in A,B; 1 mm in C. [Color figure can be viewedin the online issue, which is available at wileyonlinelibrary.com.]

Fig. 9. Effects of high KCl, TTX, and BCC treatment and cerebellar granule cell feeders on X-galstripe patterns in organotypic cultures of 1NM13 cerebella. Cerebella were dissected from 1NM13mice at P0 and cultured for 3, 5, and 10 DIV in the presence of 30 mM KCl (A), 1 lM TTX(B), or 10 lM BCC (C) or on a cerebellar granule cell feeder layer (D). After organotypic cultur-ing, cerebella were stained with X-gal. Scale bar 5 0.5 mm. [Color figure can be viewed in theonline issue, which is available at wileyonlinelibrary.com.]

sagittal zonal organization of Purkinje cells in the devel-oping cerebellum. Transgene expression could beobserved at the level of single Purkinje cell nuclei overthe surface of a whole-mount X-gal-stained cerebellumbecause of nuclear accumulation of nls-lacZ enzyme ac-tivity. The major X-gal staining patterns of 1NM13mouse cerebella are reminiscent of, but not identical to,the stripe patterns defined by immunohistochemistry forzebrin II. Nuclear X-gal staining allowed us tentativelyto assign whole-mount X-gal positive stripes as Tg11,Tg2(a, b)1, Tg3(a, b)1, Tg4(a, b)1, Tg5(a, b)1, Tg6(a,b, c)1, and Tg7(a, b)1, where a, b, and c are substripes.We also assigned three substripes Tg8(a, b, c)1 to theparafloccular compartments. These results suggest thepresence of both similar and distinct expression regula-tion between the transgene and zebrin II.

Developmental analysis of 1NM13 mouse cerebellaindicated that the major pattern of IP3R1-nls-lacZ trans-gene expression was already present by E15. 1NM13 X-gal staining patterns became indistinguishable as a resultof progressively increasing expression levels of the trans-gene in almost all Purkinje cells around the secondpostnatal week. These results indicate that the transgeneexpression pattern in 1NM13 mice can be categorizedinto the early-onset group, including L7/pcp2-lacZ(Smeyne et al., 1991; Oberdick et al., 1993; Ozol et al.,1999), OMP-lacZ (Nunzi et al., 1999), cadherin, and cal-bindin, which show stripy expression patterns beginningas early as E15 (Baader et al., 1999). The overall organi-zation of compartments revealed by L7/pcp2-lacZ, OMP-lacZ, and IP3R1-nls-lacZ transgenic mice is similar inembryos and neonates, but there are differences in thespatiotemporal patterns and X-gal staining intensitybetween them (see, for example, Figs. 3, 4 of Ozolet al., 1999; Fig. 3 of Nunzi et al., 1999; Figs. 4, 6 ofthe present study). Although the present study did notshow whether these differences reflect fundamental fea-tures of cerebellar compartmentation, many previousstudies have indicated that the PCL is organized intomultiple stripes marked with combinatorial expressionpatterns of many molecules including zebrin, phospholi-pase Cb4, heat shock protein 25, and human naturalkiller antigen 1 (for reviews see Sillitoe and Joyner,2007; Apps and Hawkes, 2009).

It is notable that the PCL at P5 had narrow gapsbetween Purkinje cell arrays stained by X-gal and/oranti-IP3R1 antibody. Within these PCL gaps, a streamof small granule cells (immunopositive for the orphannuclear receptor ROR-alpha; data not shown), from theML to the IGL was observed. These PCL gaps resemblegranule cell raphes or ribbons through which a stream ofgranule cells is thought to migrate toward the IGL(Arndt et al., 1998; Luckner et al., 2001). Raphes havebeen suggested to correlate with the boundaries ofexpression domains of cell-recognition molecules,including cadherins (Arndt et al., 1998; Luckner et al.,2001; Redies et al., 2002) and Eph-ephrins (Karamet al., 2000, 2001). Boundary formation of X-gal stripesseen in 1NM13 mice may be related to the expression

of such cell recognition molecules in the early stages,although comparative analysis of these spacing patternsremains to be performed. We also observed some gran-ule cell migration paths that are not congruent with thestripe boundaries of 1NM13 cerebella, suggesting thattransgene expression in 1NM13 mice reveals only a frac-tion of such boundaries.

Previous studies utilizing chemical lesions and axot-omy (Leclerc et al., 1988), transplantation (Wassef et al.,1990), and organotypic cultures under various conditions(Seil et al., 1995) have led to the hypothesis that theparasagittal stripes of zebrin expression are formed andmaintained independently of the extracellular milieu,afferents, neuronal activity, and/or neuron–glia interac-tions but are, rather, an intrinsic, cell-autonomous prop-erty of Purkinje cells. Similarly, the fate of Purkinje cellcompartments displayed by 1NM13 X-gal staining isprobably determined by E15. The 1NM13 expressionpatterns consist of a small number of simple Purkinje cellclusters in the early stages and become more elaborate interms of the number of clusters, their shapes, and thepositive–negative cell ratios within clusters as cerebellardevelopment advances. The next questions concern howthe subsequent lobule-specific stripe patterning in thelate stages is regulated and whether the increases in di-versity are already defined in embryonic Purkinje cells.Our cerebellar culture experiments indicated that neitherafferent projections nor membrane excitability is crucialfor the regionalized transgene expression in the 1NM13cerebellum after E15. These results suggest that the mat-uration of stripe patterns is largely dictated by intrinsicdeterminants.

The nls-lacZ transgene is driven by the 50-flankingpromoter regions (nucleotides –528 to 1169) of themouse IP3R1 gene, within which putative consensussites for transcriptional regulation have been identified(Furutama et al., 1996). The transgene in the 1NM13line was expressed predominantly in Purkinje cells, as isthe parental IP3R1 gene, in the cerebellum. Our in vitrotranscription analysis suggested that an E-box (from nu-cleotides –334 to –318) might be a candidate site forup-regulation of the IP3R1 gene in Purkinje cells viabinding to a NeuroD-related basic helix-loop-helix tran-scription factor (Konishi et al., 1997, 1999). The trans-gene in 1NM13 lines shows heterogeneous expressionpatterns in Purkinje cell subpopulations from an embry-onic stage to the second postnatal week and exhibitshigh level expression patterns in almost all Purkinje cellpopulations (Furutama et al., 1996). In L7/pcp2-lacZtransgenic mice, mutations of the putative POU tran-scription factor-binding site were reported to change theparasagittal stripe patterns of transgene expression (Ober-dick et al., 1993). From this point of view, it is of inter-est that an AT-rich/POU-binding site is also predictedin the IP3R1 promoter region (from nucleotides –505 to–476: Furutama et al., 1996), although the L7/pcp2-lacZtransgene, but not the IP3R1-nls-lacZ transgene, contin-ues to be expressed in the stripe patterns at P140 (Ozolet al., 1999). In the OMP-lacZ transgene, the OMP pro-



moter contains two sites for binding to the helix-loop-helix transcription factor Olf1/early B-cell factor (Ebf),O/E-1 site TCCCC(A/T)NGGAG (Kudrycki et al.,1993) or TCCYYRRGGAG (Wang et al., 1993), whichwas suggested to be linked to the evidence that a defi-ciency of Ebf2, one of three Ebf family members, per-turbed Purkinje cell compartmentalization (Croci et al.,2006). It might be of interest that the IP3R1 promoterhas two sequences, GCTCCAGGACA (between –422and –412) and GCTTCAAAGTG (between –9 and12), that are partially homologous to the O/E-1-likesites (Vassalli et al., 2002; Rothman et al., 2005; Micha-loski et al., 2006). Alternatively, the uneven transgeneexpression in 1NM13 probably is attributable to theinsertion location of the transgene on the chromosome.For example, a specific enhancer element, which confersstripe expression, neighboring the insertion site, mighthave been trapped in the 1NM13 line (Bonnerot et al.,1990). It is also hypothesized that a trapped enhancer orsilencer may enhance or silence (or recall) an intrinsicproperty of the IP3R1 promoter to be expressed in asubset of Purkinje cells, because the IP3R1 promoter hasvarious transcriptional regulatory elements to control thewidespread expression in many cell types at different lev-els (Furuichi et al., 1993).

In conclusion, we have established a new mousemodel with which to study the compartmentalization ofcerebellar Purkinje cells. 1NM13 mice allow us to detectclearly single-positive Purkinje cells over the surfaces ofwhole-mount X-gal-stained cerebella, because theIP3R1-nls-lacZ transgene encodes a nuclear b-gal activ-ity, a result of which is that X-gal enzymatic reactionproducts are accumulated inside the nuclei. The overallorganization of X-gal-positive stripes revealed in the1NM13 line has both similarities to and differences fromthat of the immunoreactivity for the late-onset markerzebrin II and the expression pattern of the early-onsetmarker L7/pcp2-lacZ transgene, although detailed com-parative analyses of these stripe markers are still needed.Zebrin compartments have been demonstrated to corre-late with physiological maps on the cerebellar cortexrepresenting responses to tactile (Chockkan and Hawkes,1994; Hallem et al., 1999) or electrical (Chen et al.,1996) stimulation. Thus, the heterogeneous expressionof the IP3R1-nls-lacZ transgene in Purkinje cell subsetsmay be useful as a molecular indicator of functionalunits, such as microcomplexes, of the cerebellar circuitry(Ito, 2001).

ACKNOWLEDGMENTS

We thank Dr. Hiroyuki Yoneshima of Osaka Uni-versity for valuable discussions and Dr. Roy Sillitoe ofUniversity of Calgary (present address: Albert EinsteinCollege of Medicine) for technical help.

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Expression of the IP3R1 promoter-driven nls-lacZ transgene in Purkinje cell parasagittal arrays of developing mouse cerebellum