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Molecular and Cellular Neuroscience 17, 611–623 (2001)

doi:10.1006/mcne.2001.0966, available online at http://www.idealibrary.com on MCN

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Juxtaposition of CNR Protocadherins and ReelinExpression in the Developing Spinal Cord

Patrick Carroll,1 Odile Gayet, Christian Feuillet,Sacha Kallenbach, Beatrice de Bovis,Keith Dudley, and Serge AlonsoINSERM U382, Developmental Biology Institute of Marseille (IBDM), CNRS/INSERM/Universite de la Mediterranee/AP de Marseille, Campus de Luminy, Case 907,13288 Marseille Cedex 09, France

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The CNR (cadherin-related neuronal receptors) family ofprotocadherins is of great interest because of their po-tential roles as molecular tags in the formation of specificsynaptic connections, and as receptors for reelin, duringneuronal migration, and cell body positioning. In order toknow more about potential functions of CNRs we havemapped their expression during mouse nervous systemdevelopment and compared their expression with that ofreelin and its intracellular effector Dab1 in several tissues.In spinal cord, CNRs and Dab1 are expressed in motoneu-rons, while reelin is located in adjacent cells. In the hind-brain, there is a differential expression of CNRs and Dab1in various motor nuclei. In the retina and olfactory system,we observe CNR and reelin expression but not that ofDab1. These results provide new insights into the poten-tial functions of CNRs and their possible integration in thereelin pathway during development.

INTRODUCTION

Cadherins are membrane-bound proteins thatundergo calcium-dependent homophilic interactionsthrough characteristic peptide motifs called cadherinectodomains. Classical cadherins are defined as trans-membrane molecules composed of an extracellular do-main containing five repeated cadherin motifs, calledcadherin ectodomains (EC), and an intracellular do-main which interacts with actin via the molecule b-cate-nin (Shapiro et al., 1995). In contrast, the nonclassicaladherins have 6 or more ectodomains in the extracel-ular part and intracellular domains, which differ from

1 To whom correspondence and reprint requests should be ad-ressed. Fax: (133) 4 91 26 97 57. E-mail: carroll@ibdm.univ-mrs.fr.

1044-7431/01 $35.00Copyright © 2001 by Academic Press

ll rights of reproduction in any form reserved.

hose of the classical cadherins (Yagi and Takeichi,000). Proteins containing cadherin repeats have beenmplicated in a diverse array of processes including celldhesion, control of cell division and differentiation,igration, axon pathfinding, formation of boundaries

etween tissues, synapse formation, and synaptic plas-icity. More different members of the cadherin familyre expressed in brain than in any other tissue and it ishis diversity of expression which makes the cadherinsandidates for molecules involved in patterning theervous system and specificity of synapse formationreviewed in Shapiro and Colman, 1999).

Protocadherins constitute a subfamily of cadherinshat contain 6 extracellular ectodomains. A novel familyf protocadherins was recently isolated by virtue of theact that they interact with the tyrosine kinase fyn (Koh-

ura et al., 1998). These proteins are called cadherin-elated neuronal receptors (CNRs) and are widely ex-ressed in the adult brain at synapses. Analysis of 8embers of the CNR family showed that the N-termi-

al portions of the proteins were similar but distincthereas the C-terminal portions were identical. EachNR has six ectodomains, called EC1–EC6, and puta-

ive calcium-binding sequences are present in EC2–C5. Recently, the genetic organisation of the sequencesncoding these proteins has become clear by the anal-sis of members of three families of protocadherinsPcdh a, b, and g), which are clustered in the same

region (5q31) of human chromosome 5 (Wu and Mania-tis, 1999). The mouse CNR protocadherins are the or-thologues of the human Pcdh-a family. Comparison ofcDNA sequence data with the sequence of the human

genomic locus shows that for the a and g families, thesegenes have an unusual organization. For the a family,

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FIG. 1. (A) Organization of the CNR proteins and mRNAs. CNR proteins are composed of 6 cadherin-like ectodomains (ECs), a transmembraneegion (TM), and a cytoplasmic domain. The “variable” regions are encoded by fifteen 59-exons that are spliced to three common 39-exons

containing the cytoplasmic “constant” region (Kohmura et al., 1998; Wu and Maniatis, 1999). The rat E8 clone was isolated from a ventral spinalcord cDNA bank. The mouse probe was generated by PCR from the mouse cDNA library. The rat and mouse in situ hybridization probes gaveidentical results. (B) Expression of CNRs in the E12.5 mouse embryo. Sagittal section through an E12.5 embryo. Hybridization with a DIG-labeledCNR constant region probe. Expression is observed in telencephalon (tel), olfactory epithelium (oe), hindbrain (hb), dorsal root ganglia (drg),and spinal cord (sc). Scale bar, 0.6 mm. (C) Transverse section through the head of an E14.5 embryo. The CNR probe labels cells in the spinalcord (sc), superior cervical sympathetic ganglion (scsg), the petro-nodose fused (IX and X) sensory ganglia, the facial (VII) and trigeminal (V)ganglia, as well as in the olfactory epithelium (oe) and in the retina (ret). (D) Analysis by Northern blotting of CNR expression in embryonic andadult mouse tissues. Poly(A1) RNA (5 mg/lane) was isolated from total RNA from the indicated tissues, separated by gel electrophoresis,blotted, and hybridized with a constant region-specific radioactive probe. After exposure to X-ray film, the blot was rehybrized with a GADPHprobe as a control for the quantity and integrity of RNA loaded. Two bands are visible in the region of 5.5 kb, the expected size of CNR mRNAs.

In accordance with ISH results, CNRs were expressed only in nervous system tissues. The migration positions of 28S RNA (4.7 kb) and 18S RNA(2.2 kb) are indicated.

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613Expression of CNR Protocadherins in Embryonic Spinal Cord

15 exons, each encoding the N-terminal and transmem-brane domains of one individual protocadherin, arelocated upstream from 3 exons which code for theintracellular domain of all members of the family. Dur-ing gene expression each of the N-terminal exons isspliced individually to the three intracellular exons togenerate a particular mRNA. Recently it has beenshown that this unusual organization is also found inthe mouse (Sugino et al., 2000) and is a general featureof Pcdh genes (Wu and Maniatis, 1999). Comparisonsbetween the organisation of the CNR genes and theimmunoglobulin genes have lead to speculation thatCNR gene expression may involve DNA rearrange-ments, but currently there is no evidence for this.

The function of the CNR proteins has yet to be estab-lished. The idea of a role at the synapse was suggestedby the use of a monoclonal antibody raised against anextracellular epitope specific to CNR1. Staining withthis antibody was detected at synapses in the adultneocortex suggesting that this CNR at least may be asynaptic membrane protein (Kohmura et al., 1998). AllCNRs so far tested show a very similar pattern ofexpression in the adult, where the neocortex, hip-pocampus, cerebellum, and olfactory bulb give thestrongest signals by in situ hybridization (Kohmura etal., 1998). It appears that individual neurons expressdifferent CNRs, at least in the olfactory bulb, suggestingthat these proteins could act as functional markers onneurons or at synapses.

Interest in the role of the CNRs in neuronal function hasrecently been further stimulated by the observation thatthey are receptors for reelin (Senzaki et al., 1999). Reelin isa large secreted extracellular matrix protein that interactswith several cell surface molecules including VLDLR(very low density lipoprotein receptor), ApoER2 (Apoli-poprotein E Receptor 2) (D’Arcangelo et al., 1999) and theintegrin a3b1 (Dulabon et al., 2000) and is produced in themarginal zone of the developing neocortex by specializedcells called Cajal-Retzius cells. Mice lacking reelin (reelermice) show abnormalities in cell migration and cell posi-tioning in several laminated structures in the central ner-vous system, including the cerebral cortex, hippocampus,and cerebellum (D’Arcangelo et al., 1995), a phenotypemimicked by knock-out mice lacking both VLDLR andApoER2 (Trommsdorff et al., 1999). The intracellular sig-nalling pathway activated by reelin involves the cytoplas-mic adaptor molecule Dab1, and Dab1 knock-out miceshow a phenotype indistinguishable from that of the reelermutant (Howell et al., 1997). An interaction between thereelin protein and CNR1 has been demonstrated by coim-

munoprecipitation of the two proteins by anti-CNR anti-bodies, by equilibrium binding studies and by the colo-

es

FIG. 2. CNRs are expressed in spinal motoneurons. (A) Expressionof CNRs detected on a transverse section of E12.5 mouse spinal cord(sc) at forelimb-innervating level by in situ hybridization. Ventrolat-eral cells strongly express CNR mRNAs. Strong staining is also visiblein many, but not all, cells in the dorsal root ganglia (drg). Scale bar, 70mm. (B) In situ hybridization using a DIG-labeled CNR constantregion probe was carried out on sections of E12.5 mouse embryos(blue staining). Subsequently the sections were processed for immu-nohistochemical detection of the nuclear-localized homeoproteinsIslet-1/2 by the DAB reaction (orange staining). Strongly CNR-positive cells are also Islet-positive (white arrow). Some Islet-positive motoneurons (mn) showed only weak expression of CNRs(black arrow). Many dorsal root ganglion (drg) neurons weredouble-positive. Vsc, ventral spinal cord. Scale bar, 100 mm. (C)

xpression of CNRs in transverse sections of the E14.5 mousembryo at the forelimb-innervating level. Expression has becomeore widespread than at E12.5 but remains stronger in ventrolat-

ral regions where motoneurons are situated (black arrows). Dor-al root ganglia (drg); sympathetic ganglia (sg). Scale bar, 70 mm.

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calization of staining in cortical regions using anti-reelinand anti-CNR antibodies (Senzaki et al., 1999). Altogetherhe above results point to a potential role of CNRs ineuronal migration or cell body positioning.During an in situ hybridization screen to isolate genes

xpressed in subpopulations of rat motoneurons duringevelopment, we identified a member of the CNR fam-

ly of protocadherins. Sequencing showed that thislone contains the 39 region of a rat homologue of theouse CNR family (Kohmura et al., 1998). In the mouse

mbryo strong CNR expression was detected in mostrain regions as well as in the sensory ganglia, in theetinal ganglion cells and in the olfactory epithelium.sing in situ hybridization with a CNR probe followedy staining with an anti-Islet-1 antibody, which stainsotoneurons, we show for the first time that CNRs are

trongly expressed in embryonic spinal motoneuronsnd with a pattern that is complementary to that ofeelin. The expression of CNRs and reelin in neighbour-ng cell populations in the spinal cord suggests thatNRs and reelin may interact to control some aspects ofotoneuron development.

RESULTS

Expression of CNRs Is Restrictedto the Nervous System

To date, the expression of CNRs has been demon-strated in the postnatal mouse brain (Kohmura et al.,1998) and in the E15 cortical plate (Senzaki et al., 1999).We studied the expression patterns of CNRs duringdevelopment of the nervous system. We used DIG-labeled riboprobes either covering the constant regionand a small part of the variable region from rat cDNAor a part of the mouse constant region generated byPCR (see Experimental Methods and Fig. 1). Resultswere indistinguishable. In situ hybridization on coronalsections through E12.5 and E14.5 mouse embryosshowed that CNRs are widely expressed in the nervoussystem (Figs. 1B and 1C), where neurons are undergo-ing terminal differentiation. Strong expression was seenin all sensory and sympathetic ganglia, and in the spi-nal cord. At E12.5, strong expression is observed inregions containing postmitotic neurons in the telen-cephalon, diencephalon, and hindbrain (Fig. 1B). Onsections through the E14.5 hindbrain, the CNR proberevealed expression in the superior cervical sympa-thetic ganglion (scsg), the petrosal-nodose fused (IX and

X) sensory ganglia, the facial (VII) and trigeminal (V)ganglia, as well as in the olfactory epithelium (oe) and

the ganglion cell layer of the retina (Fig. 1C). Althoughthere was strong expression in DRGs at E12.5, we sawno evidence of expression of CNRs in migrating neuralcrest derivatives at earlier stages.

These results were confirmed by Northern blot anal-ysis (Fig. 1D). Using poly(A1) RNA isolated from the

rain, limb, and spinal cord of E14.5 embryos, and fromdult cortex, cerebellum, and liver, two bands of about.5 kb were observed in nervous system tissues but notn limb nor adult liver. These two bands most probablyorrespond to the splice variants differing by 480 basesbserved by Sugino et al. (2000).Thus, expression of CNR family members is re-

tricted to the nervous system and includes nonlami-ated structures such as sensory and sympathetic gan-lia. Furthermore, the regions where CNRs arexpressed contain neurons that are post-mitotic and arendergoing terminal differentiation.

NR Family Members Are Expressed in Spinalotoneurons during Development

Strong expression of CNRs was detected in the spinalord at E12.5 at limb levels, with the strongest labelingn the ventrolateral regions, suggesting that the CNRenes are expressed in motoneurons, whereas no ex-ression was seen in the ventricular zone (Fig. 2A). Thisas confirmed by combining in situ hybridization forNR with immunostaining for the motoneuron marker

slet-1/2 (Fig. 2B). Interestingly, not all Islet-positiveells were strongly CNR-positive, suggesting that CNRsay be strongly expressed in subpopulations of mo-

oneurons. Expression of CNRs in motoneurons wasurther confirmed by RT-PCR on RNA isolated fromurified mouse motoneurons using CNR specific prim-rs (data not shown). On sections from the forelimbevel of E14.5 and E16.5 embryos, CNR expression per-isted in the spinal cord, but was still more stronglyxpressed in motoneurons (Fig. 2C, data not shown for16.5).

omparison of CNR Expression with That ofembers of the Reelin Signalling Pathway

uring Motoneuron Development

CNRs have been shown to be receptors for reelin inhe developing cortex, and reelin induces phosphoryla-ion of the cytoplasmic adaptor protein Dab1 throughts interaction with CNR proteins (Senzaki et al., 1999).

Recently it has been shown that reelin signalling is

involved in the dorsal migration of preganglionic auto-nomic motoneurons between E12.5 and E14.5 in the

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615Expression of CNR Protocadherins in Embryonic Spinal Cord

spinal cord during mouse development (Yip et al.,000). To investigate a potential role of reelin signallinghrough CNRs in the embryonic spinal cord, we com-ared the spatiotemporal expression pattern of CNRs,eelin, and Dab1 in the developing spinal cord andindbrain.Spinal cord. In situ hybridization was carried out

with each probe on serial sections of mouse spinal cordat various stages of development. Expression of CNRswas first observed in motoneurons at embryonic day10.5 (Figs. 3A and 3B). Dab1 expression also appearedat E10.5 and was colocalized with Islet-positive mo-toneurons (Fig. 3C). However, groups of Dab1-negativemotoneurons are visible. Reelin is expressed in the spi-nal cord from E9.5 onward (Ikeda and Terashima,1997). We first observed reelin expression in the spinalcord at E10.5 in a thin band of lateral cells dorsal to theregion containing motoneurons (data not shown). AtE12.5 at the forelimb-innervating level, CNRs and Dab1are expressed by motoneurons (Figs. 3E and 3F). Aspreviously reported (Yip et al., 2000), reelin is stronglyexpressed in cells dorsal to, and seemingly surround-ing, the Islet-positive motoneurons (Fig. 3G). Usinga DIG-labeled in situ probe for reelin followed by Is-let immunohistochemistry, it is apparent that reelinexpression is generally excluded from the ventrallylocated Islet-expressing motoneuron population inE12.5 embryos (Figs. 3H and 5). At the thoracic level atE13.5 we observe strong CNR expression in the ventralspinal cord (Fig. 3I), while Dab1 expression was stron-gest in the intermediolateral region where pregangli-onic neurons were revealed by NADPH-diaphorase ac-tivity (Figs. 3J and 3K), which identifies preganglionicautonomic neurons in the spinal cord (Wetts et al.,1995).

Motoneurons are arranged in columns along the ros-trocaudal axis of the spinal cord (Tsuchida et al., 1994).By whole-mount in situ hybridization using an Islet-1probe on dissected embryonic mouse spinal cord, it ispossible to visualise the motor columns along the ros-trocaudal axis after flat-mounting (Yamamoto et al.,1997). Expression of Islet-1, CNRs, Dab1, and reelin atE12.5 are compared in Fig. 4. Here we observe thatCNRs (Fig. 4B) are expressed in most of the Islet-1-positive regions (Fig. 4A) of the spinal cord. Dab1 ex-pression is absent in the medial motor column (MMC)in the thoracic region (Fig. 4C). At E12.5, reelin expres-sion is restricted to a longitudinal band along the length

of the rostrocaudal axis that seems to divide in two atthe most rostral part (Fig. 4D).

To better understand the staining pattern seen onflat-mounted preparations, vibratome sections weremade from the flat-mounted material. Spinal cordswere aligned side-by-side before embedding, such thatstaining patterns from equivalent rostrocaudal levelscould be compared. A series of sections through theforelimb-innervating region is shown in Fig. 5. Fromthis perspective, CNR expression partially overlaps thatof Islet-1, with more prominent CNR expression in themedial part of the motoneuron population. By contrast,Dab1 expression appears in the more lateral motoneu-ron population. As in Figs. 3D–3H, reelin expression isdorsal and medial to that of Islet-1, CNRs, and Dab1.Islet-1 expressing motoneurons appear to fit exactlyinto the region devoid of reelin expressing cells. Theposition of reelin expressing cells suggests that it isexpressed in subpopulations of ventral interneurons. Inmore caudal regions of the spinal cord viewed in flatmount, because ventral columns obscure deeper-lyingdorsal columns, reelin-positive cell populations appearas a single longitudinal column (Fig. 4D). Rostral to theforelimb, one of the columns is displaced laterally. Theidentity of these reelin expressing cell populations re-mains to be elucidated.

In summary, in the spinal cord, as in the developingneocortex, reelin and CNRs are expressed in adjacentcell populations. Motoneurons that express CNRs oftenbut not always express Dab1. CNR and Dab1 express-ing motoneurons first migrate to the reelin-negativeventral spinal cord with Dab1-positive preganglionicneurons later migrating dorsally but not entering thezone where reelin is expressed (Yip et al., 2000).

Hindbrain. In the developing hindbrain, we ob-served a more complex pattern of expression of thesegenes. Hybridization in situ using an Islet-1 probeallows the visualisation of the various motor nuclei.At E12.5, CNR expression is weak and diffuse in thehindbrain with the exception of expression in thetrigeminal (Vth) and facial (VIIth) (Fig. 4B). In con-trast with the result for the CNR probe, the Dab1probe strongly labels the facial (VIIth) nucleus as wellas a broad band of unidentified cells rostral to thisstructure and weakly the trigeminal (Vth) nucleus(Fig. 4C). However, the hypoglossal motoneurons(XIIth) were negative for Dab1. Reelin expression lagsbehind that of Dab1 (Fig. 4D) and is not present in thevicinity of strongly-labeled Dab1-expressing facialmotoneurons. The expression of CNR and reelin is

dynamic as evidenced by the patterns seen at E14.5(Figs. 4F and 4H). CNR expression is observed in the

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616 Carroll et al.

facial nucleus and diffusely throughout the hindbrain

FIG. 3. Expression of CNRs, Dab1, and reelin in the developing spinamouse embryos. (A, B) CNR expression is found in the same region ahybridization (blue) followed by Islet-1/2 immunohistochemistry (orangDab1-negative motoneurons are also evident. (D–G) Comparison of thea series of sections from an E12.5 mouse embryo through the forelimbincluding Islet-positive motoneurons (framed by dotted line). (F) Dab1 ethe most lateral cell populations. (G) Reelin expression is dorsal and mexpression of reelin and Islet-1 in the ventral spinal cord. Reelin in situ hy(I–K) Expression of CNRs and Dab1 at the thoracic level at E13.5. (I) CNDab1 is expressed strongly in the region where dorsally migrating autonvisualised using a histochemical reaction for NADPH-diaphorase activi

(Fig. 4F). Bands of unidentified reelin expressing cellscan be seen rostral and caudal to the facial nucleus at

E14.5 but the facial nucleus itself is negative (Fig. 4H).

. (A–C) CNRs and Dab1 are expressed in spinal motoneurons in E10.5t-1 positive motoneurons at E10.5 (adjacent sections). (C) Dab1 in situouble-labeled cells are visible within the motoneuron population. Manyssion of CNRs, Dab1, and reelin and the motoneuron marker Islet-1 onrvating region. (E) CNRs are expressed strongly in latero-ventral cellsssion is more restricted than that of CNRs and appears to be limited toto CNR and Dab1 expressing cell populations. (H) Mutually exclusivezation (blue) was followed by Islet-1/2 immunohistochemistry (orange).re strongly expressed in the most ventral regions of the spinal cord. (J)preganglionic neurons are located. (K) Preganglionic neurons (pg) wereJ, K are adjacent sections. Scale bars, 100 mm.

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The facial nucleus continued to express Dab1strongly at this later stage (Fig. 4G).

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617Expression of CNR Protocadherins in Embryonic Spinal Cord

CNR Expression in the Olfactory andVisual Systems

Cells in the retinal ganglion cell layer of the retina

FIG. 4. Expression patterns of molecules implicated in reelin signal(A–D) Flat-mounted preparations of spinal cords and hindbrain fromindicated DIG-labeled probes. The spinal cord was cut along the dorcentre (“open book” preparation). Islet-1 labels most motoneuron popnuclei and the medial motor column (MMC) in the spinal cord areevident (A). (B) CNRs are expressed in cells in the ventral spinallimb-innervating motoneurons, the trigeminal (Vth) motor, and weakVIIth (facial nucleus) strongly and the Vth (trigeminal motor) nucleuscord, Dab1 labels laterally located motoneuron populations at the limbof cells along the rostrocaudal axis anteriorly as far as the hypoglmotoneurons in the spinal cord. Scale bar, 400 mm. (E–H) Mouse E14is seen in the facial nucleus. (G) At this stage, Dab1 expression remainrostrally. The hypoglossal (XIIth) nucleus is negative. (H) Reelin expthe facial nucleus.

were CNR-positive as were presumptive olfactory neu-rons in the olfactory epithelium (Fig. 1C). Thus we

decided to look at these structures in more detail withrespect to CNR, reelin, and Dab1 expression.

Olfactory system. As already described, CNR-pos-

by whole-mount in situ hybridization on spinal cord and hindbrain.mouse embryos of the same litter after hybridization in situ with the

idline and flat-mounted such that the ventral midline appears at thens: the Vth (trigeminal motor), VIIth (facial), and XIIth (hypoglossal)d by dotted lines. The migration pathway of facial motoneurons isincluding the medial motor column (MMC), the area containing

the hypoglossal (XIIth) and facial (VIIth) nuclei. (C) Dab1 labels thely. The hypoglossal (XIIth) nucleus is negative for Dab1. In the spinall. The MMC is negative. (D) Reelin is expressed in longitudinal bands(XIIth) nucleus. Reelin-positive cells are dorsal to Islet-expressing

ndbrain. (F) CNR expression becomes more widespread. Expressionng in the facial nucleus and in a band of unidentified cells extendingn is present in the hindbrain at E14.5. No expression is observed in

lingE12.5sal mulatioframecordly inweakleve

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itive cells were observed in the olfactory epithelium.The axons of olfactory neurons in the olfactory epithe-

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618 Carroll et al.

lium form the olfactory nerve that grows into the rostraltelencephalon at E12.5 in the mouse, at the same time asthe olfactory bulb primordium begins to develop (Gongand Shipley, 1995). In addition to cells in olfactoryepithelium, the CNR probe detected at E12.5 a strongly-labeled population of apparently migrating cells be-tween the olfactory epithelium and the rostral telen-cephalon (Fig. 6A). These cells are possibly migratingluteinizing hormone-releasing hormone (LHRH) neu-rons that arise from the olfactory placode and migratealong the vomeronasal nerve to the forebrain (Yoshidaet al., 1999, and refs. therein). Expression of CNRs wasalso detected in the vomeronasal organ (Jacobson’s or-gan) (not shown). We then looked at reelin expressionin the same tissues (Fig. 6B). Reelin expression is strongin Cajal–Retzius cells of the marginal zone of the telen-cephalon. Weak reelin expression is visible in the regionbetween the olfactory epithelium and the telencepha-lon. A similar expression was observed by Ikeda andTerashima (1997) and was suggested to represent ex-pression by cells along the olfactory nerve. The identityof these cells was not determined. As expected, Dab1 isexpressed in the telencephelon (Fig. 6C). However, onlyweak diffuse staining by the Dab1 probe was seen in thetwo other strongly CNR-positive tissues: the olfactoryepithelium, and the apparently migrating cells betweenthe olfactory epithelium and the telencephalon.

Retina. In the E12.5 retina, strong CNR staining isseen in the ganglion cell layer, where primitive retinalganglion neurons (rgc) are differentiating (Fig. 6D), whilereelin is expressed in the retinal pigment epithelium anddiffusely in the main part of the retina (Fig. 6E). At E14,reelin expression is also strong in the ganglion cell layer(data not shown; Schiffman et al., 1997). No Dab1 expres-sion was detected in the embryonic retina (Fig. 6F), con-firming previous observations (Rice and Curran, 2000).

DISCUSSION

Our results show that the CNR family of protocad-herins is expressed widely in the nervous system dur-ing mouse development. Potentially important sites ofexpression include the embryonic spinal cord, sensoryand sympathetic ganglia, retinal ganglion cells and theolfactory epithelium. No embryonic tissues outside thenervous system express CNR genes. We have shown forthe first time that CNRs, and the adaptor moleculeDab1, are expressed in motoneurons in the spinal cordand hindbrain in a pattern complementary to that of

reelin. Although this is reminiscent of the expressionprofile of these three molecules in the cortex (Senzaki et

1i

al., 1999), in the hindbrain CNR/Dab1 expression pre-cedes that of reelin.

CNRs Are Expressed in Neurons duringMigration and Synaptogenesis

For classical cadherins, homophilic interactions areknown to activate intracellular signalling pathways(Shapiro et al., 1995). We find expression of CNRs inmotoneurons, sensory and sympathetic ganglia at atime when these neurons are forming synapses withtheir targets and receiving synapses from their affer-ents. The previous demonstration at pre- and postsyn-aptic sites of CNRs would be consistent with a role forhomophilic interactions between these molecules insynapse formation and maintenance (Kohmura et al.,998). Thus CNRs could potentially be implicated inynapse formation between DRG neurons and spinaleurons, autonomic preganglionic neurons and sympa-

hetic neurons, spinal interneurons and motoneurons.he absence of CNR expression in developing musclerecludes a role for CNRs via homophilic interactionst the neuromuscular junction. Similarly, the timing ofxpression of CNRs in retinal ganglion cells and olfac-ory neurons in the olfactory epithelium would be con-istent with a role in axon guidance/synaptogenesis inhese neurons.

In their study on the adult brain Kohmura and col-eagues used probes specific for individual members ofhe CNR family to demonstrate that different sets ofeurons probably express different sets of CNR genesKohmura et al., 1998). In this study we have used aonstant region probe which will detect with equalfficiency any of the 15 possible mRNAs encoded by theNR family. It will now be interesting to see whether

hese signals correspond to the expression of individualNRs or combinations of several isoforms in particular

ubpopulations of cells, e.g., motoneuron pools, subsetsf DRG neurons.

Potential Role for CNRs inotoneuron Migration

CNRs are also expressed by motoneurons at earliertages, when they are migrating to their final locationsn the neural tube. Current models suggest that reelinan interact with several receptor molecules at the cellurface: VLDLR/ApoER2 receptors that bind Dab1, andNRs that recruit a src-like intracellular kinase for thectivation of Dab1 by phosphorylation (Senzaki et al.,

999). We show here that CNRs and Dab1 are expressedn populations of motoneurons in the spinal cord and

sut

619Expression of CNR Protocadherins in Embryonic Spinal Cord

hindbrain from E10.5 onward, while reelin was ex-pressed in adjacent cells as previously reported (Ikedaand Terashima, 1997; Yip et al., 2000). By their position,we suppose that the reelin expressing cells are sub-populations of spinal interneurons. In the reeler mutant,as well as the Dab1 knockout mouse, neurons that arenow known to express CNRs fail to migrate normally inthe cortical region with the result that these cells fail tosplit what is called the preplate into the subplate andthe marginal zone, resulting in aberrant formation ofthe cortical laminae (Goffinet, 1984b; reviewed in Gil-more and Herrup, 2000). It has also been shown thatmotoneurons of several hindbrain motor nuclei aremal-positioned in reeler mice (Goffinet, 1984a; Fujimotoet al., 1998).

It is still unclear how reelin acts at a molecular levelin organising neural development. Several models haveproposed that reelin may act as an attractant or as arepulsive agent to migrating cells or that reelin inter-rupts the cellular interactions between migrating neu-rons and radial glial cells in the developing cortex caus-ing the neurons to detach. In common with eventstaking place during development of the cortex, the de-velopment of the spinal cord involves complex cellmigrations including both radial and tangential migra-tions (Leber and Sanes, 1995; reviewed by Hatten, 1999).Recent studies by Yip and colleagues (2000) haveshown that reelin is involved in the control of position-ing of autonomic preganglionic neurons in the spinalcord. Preganglionic neurons are a subtype of motoneu-ron that migrate radially from the neuroepithelium tothe ventrolateral spinal cord along with somatic mo-toneurons. In a secondary tangential migration thesecells then migrate dorsally to give rise to the interme-diolateral column, with some cells continuing their mi-gration to form the intermediomedial column which isfound next to the central canal (Phelps et al., 1993).Analysis of both the reeler and Dab1 knockout micehows that the majority of the preganglionic cells pop-late the intermediomedial column rather than the in-

ermediolateral column (Yip et al., 1999, 2000). Oneconclusion from these observations is that migratingpreganglionic neurons avoid areas of reelin expression(Yip et al., 2000).

Our results for the spinal cord and hindbrain showthat motoneurons expressing CNRs and Dab1 are con-fined to regions where reelin expression is absent. In thehindbrain, reelin expression appears subsequently tothat of CNR/Dab1. These results support the hypothe-sis suggested by Yip et al. (2000) for preganglionic neu-

rons that the interaction of reelin receptors on the sur-face of the motoneurons with reelin secreted by

surrounding cells acts to lock them into their positionafter arrival at their final destinations.

Our results show for the first time the expression ofDab1 in certain populations of motoneurons. The factthat several motoneuron populations known to be af-fected in the reeler mutant such as preganglionics in thespinal cord, and branchiomotor nuclei in the hindbrain,all express Dab1, suggests that Dab1 expression mightbe indicative of cells susceptible to be mal-positioned inreeler mice. It is noteworthy that the differences that wesee in the intensity of Dab1 expression in the trigeminaland facial nuclei correlates with the severity of thephenotype in reeler mice (Goffinet, 1984a). This raisesthe question whether the motoneuron populations thatwe show express Dab1 strongly in limb-innervatingregions at E12.5, where no preganglionic neurons arepresent, might also be affected in reeler mice. It isknown, for example, that the lateral region of the lateralmotor column (LMCL) is formed by the migration oflate-born motoneurons past the medial lateral motorcolumn (LMCM) under the influence of retinoic acid(Sockanathan and Jessell, 1998).

A population of cells that expressed CNRs stronglywas observed between the olfactory epithelium and thetelencephalon. These are probably Luteinising Hor-mone Releasing Hormone (LHRH) neurons migratingto the olfactory bulb: these cells show aberrant migra-tion in individuals displaying the X-linked disease Kall-mann syndrome (Kallmann et al., 1944; del Castillo et al.,1992).

Not All CNR-Positive PopulationsAre Dab1-Positive

In the spinal cord, the relative distribution of CNR-and Dab1-expressing cells depended on the positionalong the rostrocaudal axis. At the limb level expressionof CNRs and Dab1 is overlapping, while in the thoracicregion CNR mRNAs are in ventral motoneurons whileDab1 expression is associated with dorsal preganglionicneurons. In several other tissues we also see a differen-tial expression of these genes. For instance, in the hind-brain, we saw strong expression of Dab1 in the facialnucleus where CNR expression is much weaker. Incontrast, CNR expression is strong in the trigeminalnucleus where Dab1 expression is weak at E12.5. In theretina and olfactory system we observed expression ofCNRs and reelin but not Dab1. The axons of CNR-positive olfactory neurons enter the reelin-rich environ-ment of the telencephalon. Similarily the CNR-positive

LHRH neurons migrate into the reelin-rich telencepha-lon via the olfactory nerve. Whether CNR/reelin inter-

sassr

fdCt

620 Carroll et al.

actions are involved in these processes remains to beseen. Potential explanations for these results include: areelin/CNR signalling pathway that does not involveDab1; nonreelin associated CNR signalling pathways,i.e., homophilic interactions. In the latter case, CNRscould be involved in axon guidance of retinal ganglioncell axons leaving the retina and olfactory neuron pro-jections to the olfactory bulb.

In summary, we have analyzed the expression ofCNRs in the developing mouse and compared this ex-pression with that of reelin. In addition to the corticalplate in the neocortex (Senzaki et al., 1999), we show foreveral other regions of the nervous system that CNRsre expressed in neurons that are adjacent to, or areynaptic partners of reelin-expressing cells. While in

FIG. 5. Spatial relationship between reelin expressing cells and cellsexpressing Islet, CNRs, and Dab1. (A–D) Flat-mounted E12.5 spinalcords as shown in Fig. 4 were aligned longitudinally and embeddedin gelatine for vibratome sectioning. Sections (100 mm) from theorelimb-innervating region are compared. Reelin expressing cells areorsal and medial to motoneuron populations expressing Islet1,NRs, and Dab1. Dorsal (d); ventral (v); medial (m); lateral (l); ven-

ricular zone (vz).

ome cases the CNR-expressing cells are affected ineeler mice, in many other sites of CNR expression, such

as the retina and the olfactory system, are not known tobe affected. Thus it is probable that CNRs have multiplefunctions at different stages of development. Part ofthese functions may involve interactions with reelin.

EXPERIMENTAL METHODS

Construction of Rat and Mouse Spinal Cord cDNALibraries and Screening by in Situ Hybridization

Polyadenylated mRNA isolated from embryonicdays 12, 14, and 16 mouse or E14 rat ventral spinalcords was used to generate cDNA with an oligo(dT)primer. Double-stranded cDNAs were cloned in a uni-directional orientation in the ZAP Express vector usingthe Gigapack III Gold cloning kit (Stratagene). Mouseand rat primary libraries consisted respectively of 8.7and 1.4 3 106 independent clones with an average insertsize of 2 and 1.6 kb. An in situ hybridisation basedscreen was carried out by hybridising DIG-labeled ri-boprobes from individual cDNA clones to dissectedembryonic rat spinal cords as described below.Amongst several clones showing a restricted pattern ofexpression in the rat spinal cord, we identified a cDNAclone representing a rat homologue of a mouse CNRmRNA. The rat CNR clone, E8, is 3 kb long and containsthe transmembrane domain and the whole cytoplasmicdomain (Fig. 1A). The derived amino acid sequence ofthe rat partial variable region is homologous to bothorthologous sequences, mouse CNR2 (73% identicalamino acids) (Kohmura et al., 1998) and human Pcdh6(75%) (Wu and Maniatis, 1999). The protein sequence ofthe constant region encoded by the rat CNR cDNA,which includes potential fyn kinase binding sites (Koh-mura et al., 1998), was 98.7% identical to that of themouse CNR2 and 97.4% identical to that of the humanPcdh6.

Northern Blot Analysis

Total RNA was extracted from cortex, cerebellumand liver of adult mouse and also from brain, limb andspinal cord of E14 mouse embryos using Trizol (Gibco-BRL) (Chomczynski and Sacchi, 1987). PolyadenylatedmRNAs were isolated on oligo-dT cellulose using theFastTrack 2.0 kit (Invitrogen). Five micrograms of eachRNA sample were electrophoresed on 1% agarose

formaldehyde gel, transferred to Hybond N Nylonmembrane (Amersham) and hybridised to a 1-kb cDNA

(ttbse in th( bino

621Expression of CNR Protocadherins in Embryonic Spinal Cord

probe corresponding to an internal fragment of theCNR constant region. GAPDH transcript levels wereused to compare the amounts of RNA loaded on the gel.

In Situ Hybridization on Embryo Sections

Antisense digoxigenin (DIG)-labeled riboprobe forthe CNR constant region was produced from a PBK-CMV plasmid isolated from a rat E14 ventral spinalcord cDNA library. Antisense DIG (or fluorescin)-la-beled riboprobe for reelin (nucleotides 5656–7131 ac-cording to the Genebank Accession No. NM_011261)was produced from a murine cDNA pBluescript plas-mid (gift from A. Goffinet) using a DIG-RNA labellingkit (Roche Diagnostics), following the manufacturer’sinstructions. Antisense DIG-labeled riboprobe for Dis-abled-1 (Dab1) was produced from a murine cDNA in apGEM plasmid. This Dab1 cDNA plasmid was ob-tained by cloning a PCR product containing the last 245

FIG. 6. Expression of molecules implicated in reelin signalling in theA) In situ hybridization reveals CNR expression in scattered cells inhe olfactory epithelium and the olfactory bulb primordium in theelencephalon by Cajal-Retzius (C-R) cells in the marginal zone of theetween the olfactory epithelium and the telencephalon (large arrowtaining is seen in the olfactory epithelium (oe). (D–F) Mouse E12.5 rearly-born primitive retinal ganglion cells (rgc). (E) Reelin is expressedF) Little or no expression of Dab1 is evident. Note: tissue is from al

bp of the coding region plus the whole 39 UTR sequenceinto the pGEM vector (Promega). The PCR reaction was

performed directly on the mouse ventral spinal cordlibrary using the two following oligonucleotides: thesense 59-CCACACCATCTACCAACTCACC-39 and T7oligonucleotides. This Dab1 probe can detect both 555and 271 amplified transcripts (see Table 1 in Ware et al.,1997).

In situ hybridizations were performed as describedpreviously (Schaeren-Wiemers et al., 1993; Yamamoto etal., 1997) on 16-mm-thick frozen transverse sections pre-pared from E10.5–E14.5 mouse embryos fixed with 4%paraformaldehyde in 0.12% M phosphate buffer (pH7.4), and cryopreserved in 15% sucrose and 0.12 Mphosphate buffer (pH 7.2), before embedding in OCTcompound (Miles). After hybridization overnight at70°C with a riboprobe, the slides were washed twice in13 SSC, 50% formamide at 70°C for 30 min and blockedin the presence of 4% blocking reagent and 20% inacti-vated sheep serum. The slides were then incubatedwith anti-DIG-alkaline-phosphatase(AP)-conjugated

loping olfactory system and eye. (A–C) Mouse E12.5 olfactory system.lfactory epithelium (oe). Very strongly labeled cells are seen betweencephalon (tel) (large arrow). (B) Reelin is strongly expressed in thecephalon (indicated by small arrow). Lightly stained cells are found) Dab1 is expressed in forebrain by differentiating neurons. Diffuse(D) CNR mRNAs are found in the inner nuclear layer comprising thee retinal pigment epithelium (rpe) and weakly throughout the retina.

mice, thus the retinal pigment epithelium (rpe) is not pigmented.

devethe otelentelen). (C

tina.

(or anti-fluorescin-AP-conjugated) antibody (Roche Di-agnostics), washed, and revealed with NBT/BCIP stain-

oa(wP(ih

D

622 Carroll et al.

ing. Negative controls were performed on sections ei-ther with a sense probe or without probe.

Double in Situ Hybridization/Immunohistochemistry

Slides were rinsed in PBT (PBS, 0.1% Triton), andsections were incubated 1 h with blocking solution con-taining 2% BSA, 2% heat-inactivated donkey serum inPBT and then overnight at 4°C with a mixture of twoprimary monoclonal antibodies anti-Islet-1/2 (fromDev. Studies Hybridoma Bank): 4D5 (1:100) and 2D6(1:500). After three washes in PBT, slides were incu-bated 1 h at RT with the biotin donkey anti-mousesecondary antibody (1:1000) (Jackson ImmunoResearchLab.). After wash in PBS, and in TBS (Tris–HCl 50 mM,NaCl 0.15 M, pH 7.6), slides were incubated 30 min atRT in presence of the complex ABC streptavidin/HRPin TBS and revealed with DAB in the presence of H2O2.After dehydration and xylene incubation, slides weremounted in Eukitt. Negative controls were performedby omitting primary antibodies or secondary antibody.

Whole-Mount in Situ Hybridization

Whole-mount in situ hybridization (ISH) was carriedut as described by Henrique et al. (1995). Brain stemsnd spinal cords were dissected in PBS and fixed in 4%w/v) paraformaldehyde/PBT overnight at 4°C. They

ere progressively dehydrated and then rehydrated inBT-EtOH washes and then treated with proteinase K

10 mg/ml in PBT). Samples were postfixed for 20 minn 4% PFA, 0.1% glutaraldehyde in PBT, and then pre-ybridized 1 h at 70°C in 1.33 SSC, 50% formamide, 2%

Tween 20, 0.5% Chaps, 5 mM EDTA, and 50 mg/mlyeast RNA. Samples were hybridized with DIG-labeledriboprobes in the same buffer at 70°C overnight.Washes in hybridization buffer were followed byRNase A treatment (10 mg/ml in 0.5 M NaCl, 10 mMTris, pH 7.5, and 0.1% Tween 20, 30 min at 37°C). RNasewas inactivated by two washes of 30 min at 65°C inhybridization buffer. Samples were then blocked 1 h atRT in MABT (0.1 M maleate, 0.15 M NaCl, and 0.1%Tween 20, pH 7.5) containing 20% sheep serum andincubated overnight at 4°C with anti-DIG-alkalinephosphatase(AP)-conjugate (Roche Diagnostics) di-luted 1:2000 in MABT with 2% sheep serum. Afterseveral washes with MABT under gentle agitation, rev-elation was performed with NBT/BCIP (Roche Diag-nostics) in 0.1 M Tris, pH 9.5, 0.1 M NaCl, 50 mMMgCl2, and 0.1% Tween 20. After staining, the brain

stems and spinal cords were flat-mounted as open-bookpreparations in 75% glycerol, 4% PFA.

D

Vibratome sections of flat-mounted spinal cordsmade by aligning cords side-by-side before embeddingin gelatine. Transverse sections of 100 mm were cut.

NADPH-Diaphorase Histochemical Staining

NADPH-diaphorase histochemical staining of neuralpopulations indicates the presence of nitric oxide syn-thase. Staining was carried out as described in Wetts etal. (1995). Embryo sections were treated with 1% Triton-X-100 in 0.12 M Tris buffer for 15 min. Sections werestained overnight at 37°C in 0.05 mg/ml nitroblue tet-razolium (Sigma) and 0.125 mg/ml NADPH (Sigma) inTris buffer containing 1% Triton. After staining, sec-tions were washed and dehydrated in ascending con-centrations of ethanol before coverslipping in Eukittsolution.

ACKNOWLEDGMENTS

We thank members of INSERM Unite 382 for many helpful discus-sions and especially C. Henderson for comments on the manuscriptand K. Loulier for her help in ISH experiments. A. Goffinet gener-ously donated the mouse reelin probe. The two monoclonal anti-Isletantibodies (39.4D5 and 40.2D6), developed by T. M. Jessell, wereobtained from the Developmental Studies Hybridoma Bank main-tained by the Department of Pharmacology and Molecular Sciences,Johns Hopkins University School of Medicine, Baltimore, MD 21205,and the Department of Biological Sciences, University of Iowa, IowaCity, IA 52242, under Contract NO1-HD-2-3144 from the NICHD. Ourlaboratory is supported by INSERM, CNRS, the Association Francaisecontre les Myopathies (AFM), and by the European Community (ECBiotechnology Grant No. BIO4-CT96-0433). P.C. was supported by anEC fellowship from the TMR Work Programme and by a bourse fromthe Federation pour la Recherche Medicale (FRM). C.F. is a recipientof a scholarship co-funded from INSERM and the PACA region.

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Received October 26, 2000Revised January 2, 2001

Accepted January 9, 2001Published online March 30, 2001