The Journal of , September 1992, Q(9): 35253534

Restricted Expression of N- and R-Cadherin on Neurites of the Developing Chicken CNS

Christoph Redies,” Hiroyuki Inuzuka,b and Masatoshi Takeichi Department of Biophysics, Faculty of Science, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606, Japan

The expression of two cadherins, N- and R-cadherin, was intense study in recent years. A number of developmentally mapped in the CNS of chicken embryos of 6-l 1 d incubation, regulated moleculesthat show a restricted pattern of expression focusing on the sensory and motor fiber systems. In the in the developing CNS have been described. These molecules spinal cord, the laterally located fibers of the dorsal funiculus are thought to play important roles in growth cone elongation, express N-cadherin while the medially located fibers do not. axonal guidance, and fasciculation (for review, seeRutishauser These two fiber systems have a different course within the and Jessell,1988). Most of the moleculesof this kind identified CNS but associate to form the spinal dorsal roots. In the to date belong to the immunoglobulin superfamily of molecules. hindbrain, N-cadherin is expressed by the descending tri- Examples are Ll (Rathjen and Schachner, 1984), fasciclin II geminal (general somatic sensory) tract, which is contiguous (Grenningloh et al., 199 l), fasciclin III (Pate1et al., 1987), SC1 with the N-cadherin-positive zone of the dorsal funiculus of or DM-GRASP (Burns et al., I99 1; Tanaka et al., 199l), TAG- 1 the spinal cord. R-cadherin is not expressed by sensory (Dodd et al., 1988), Bravo (de la Rosaet al., 1990), and others. fibers, but is expressed by the visceral motor system of the In this study, we show that membersof another superfamily of vagus and glossopharyngeal , which are N-cadherin molecules, the cadherins (Takeichi, 1988, 1991), are also ex- negative. The motor neurites expressing R-cadherin have a pressedin the CNS in a temporally and topographically re- different course within the brain than the sensory neurites stricted pattern and may provide guidance cues for mi- expressing N-cadherin, although they form the common sen- gration. sory/motor roots of the vagus at the surface of the Cadherins are a family of moleculesmediating Ca’+-depen- brain. dent cell-cell adhesionin various tissues(Takeichi, 1988, 1991). The possibility that N-cadherin provides a guidance cue A cadherin abundantly present during early neurogenesis,N-ca- for sensory axon migration within the CNS by a homophilic dherin (Hatta et al., 1987), was shown to play a role in neuru- adhesion mechanism was investigated in vitro. Explants from lation (Hatta and Takeichi, 1986; Detrick et al., 1990; Fujimori sensory spinal ganglia expressing N-cadherin were placed et al., 1990), neuroepithelial morphogenesis(Matsunaga et al., on N-cadherin-transfected neuroblastoma cells, and axon 1988b; Takeichi et al., 1991), axon elongation (Matsunaga et outgrowth was visualized. Results showed that the sensory al., 1988a; Tomaselli et al., 1988; Bixby and Zhang, 1990; Le- defasciculate and closely follow the cell-cell bound- toumeau et al., 1990; Doherty et al., 1991), and axon fascicu- aries between transfected cells where high levels of N-ca- lation (Drazba and Lemmon, 1990). dherin are expressed. Recently, evidence for the presencein brain of several other These results show that the two cadherins, like members cadherinswas found (Inuzuka et al., 199 la,b; Napolitano et al., of the immunoglobulin superfamily of molecules, are ex- 1991; Ranscht and Dours-Zimmermann, 1991; Suzuki et al., pressed in a topographically restricted fashion during chick I99 1). The role ofthese different cadherinsin CNS development brain development. They furthermore suggest that N-cad- is unclear. In this work, westudied the expressionof N-cadherin, herin expression by neurites may play a role in guiding comparing it with that of another cadherin, R-cadherin (Inuzuka these neurites along CNS paths that express the same mol- et al., 199I a,b). We showthat, during chicken CNS development ecule. [6-l 1 d of incubation (E6-El l)], these two moleculesare dif- ferentially expressedin different fiber systems.The expression During the development of the CNS, axons grow along defined of the cadherins in specific fiber tracts suggeststhat cadherins paths to their targets, sometimesover long distances. The mo- may provide guidancecues for axon elongation. It is thus con- lecular mechanismsguiding axonal growth have been under ceivable that axons expressinga specificcadherin may be guided to elongatealong tracts expressingthe samecadherin. This hy- Received Jan. 13, 1992; revised Mar. 30, 1992; accepted Apr. 2, 1992. pothesiswas investigated in vitro by studying the pattern ofaxon We thank Dr. H. Fujisawa for his generous gift of antibodies and Dr. H. Nak- outgrowth from N-cadherin-positive sensoryneurons on neu- amura for his advice on the DiI experiments. This work was supported by research roblastoma cells expressingthe same molecule. grants from the Ministry of Education, Science and Culture of Japan. CR. was a recipient of a fellowship from the Japan Society for the Promotion of Sciences. Correspondence should be addressed to Dr. Masatoshi Takeichi, Department Materials and Methods of Biophysics, Faculty of Science, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606, Japan. Animals. Fertilized eggs from White Leghorn chickens were obtained aPresent address: Department of Biochemistry, Max Planck Institute for De- from a local farm and incubatedin a forced-draftincubator at 37°C. velopmental Biology, Spemannstrasse 35/H, W-7400 Tiibingen, Germany. Stagingof the embryoswas accordingto Hamburgerand Hamilton bPresent address: Biology Research Laboratories, Research and Developmental (1951). Division, Takeda Chemical Industries, Yodogawa-ku, Osaka 532, Japan. Generation qfmonoclonal antibodies against R-cadherin. R-cadherin Copyright Cc) 1992 Society for Neuroscience 0270-6474/92/123525-10$05.00/O fusion proteinswere prepared as previouslydescribed (Inuzuka et al., 3526 Redies et al. * Cadherin Expression in Chicken

I99 la). Balb/c mice were immunized by intraperitoneal injection of the serum and antibiotics (DHIO). These cells were cotransfected with I proteins (200 &animal) emulsified with Freund’s complete adjuvant. fig of pMiwcN, a chicken N-cadherin expression vector (Fujimori et After boosting with the same amount ofantigens emulsified with Freund’s al., 1990), and 0.1 pg of pSTneoB encoding the neomycin resistance incomplete adjuvant, splenocytes of the mice were collected and fused gene (Katoh et al., 1987) in calcium phosphate precipitates. After se- with P3UI myeloma cells. Culture supematants of the hybridomas lection with G418 as described (Hatta et al., 1988) two stable clones obtained were screened for reactivity to R-cadherin by immunoblotting. expressing N-cadherin (2a-Ncad 1 and 2a-Ncad2) were obtained. Clones Two monoclonal antibodies, termed RCD- 1 and RCD-2, were isolated, were tested for immunoreactivity with the monoclonal antibody NCD-2. and RCD-2 was used throughout the present experiments. As a control, two G4 1g-resistant clones that did not express N-cadherin Immunocytochemica/s. N-cadherin rat monoclonal antibody NCD-2 were also selected (2a-0 1 and 2a-02). (Hattaand Takeichi, 1986) and R-cadherin mouse monoclonal antibody Explant culture of sensory ganglia on N-cadherin-trarufected cells. RCD-2 (see above) were partially purified by 50%-saturated ammonium N-cadherin-transfected and control neuroblastoma clones were plated sulfate precipitation of hybridoma supematant. Anti-chicken neurofi- on glass coverslips of 2.5 cm diameter placed in 3.5 cm culture dishes, lament mouse monoclonal antibody (Hatta et al., 1987) was a generous at subconfluent density (4-7 x IO5 cells/dish). Forskolin (WakoPure gift of Dr. H. Fujisawa. Appropriate secondary antibodies labeled with Chemicals) was added to the DH 10 medium at a concentration of 0.03 FITC, biotin, or Texas Red were purchased from Amersham. Strept- mM to prevent clumping of the cadherin-transfected cells. The same avidin labeled with FITC or Texas Red (Amersham) was used to detect medium was also used for the untransfected control cultures. One day biotin-labeled secondary antibodies. after plating, the cells in each dish had grown to confluence and were Immunostainingprocedure. Immunostaining procedures were essen- used as a substrate for axon outgrowth. tially as described elsewhere (Inuzuka et al., 199 la,b). Briefly, embryonic Sensory ganglia from embryos of 6 d of incubation (E6; stage 28-29) brains were fixed in freshly depolymerized 4% formaldehyde in Hank’s were dissected under the microscope and cut into fragments of about buffered salt solution (HBSS), immersed in a graded series of sucrose 50-I 50 Frn in diameter. The fragments were placed onto the transfected solutions (12%. 15%, and 18% sucrose), embedded in TissueTek (Miles), or nontransfected neuroblastoma monolayers after the medium was and frozen in liquid nitrogen. Cryostat sections of 20-30 pm thickness replaced with fresh DHlO medium supplemented with 10 &ml of were mounted and dried on slide glasses coated with chromium potas- NGF, 2% heat-inactivated chicken serum, and forskolin. Half of this sium sulfate and gelatin. Sections were postfixed in 4% formaldehyde/ medium was replaced with fresh medium every 12 hr. Forty to 48 hr HBSS and membranes permeabihzed in -20°C cold methanol. Unspe- later. the cells were fixed in 4% formaldehvde/HBSS for 10-20 min. cific binding was reduced by blocking the sections in a solution of Tris- washed in HBSS, and stained with the anti-chicken neurolilament an- buffered saline (TBS) containing I mM Ca2+, 5% skim milk, and 0.3% tibody as described above. Triton X-100. For double-label immunostaining, sections were first In some experiments, the IgG fraction ofa polyclonal rabbit antiserum incubated with one of the primary antibodies for 1-14 hr, followed by against chicken N-cadherin, or the IgG fraction of nonimmunized con- incubation with the appropriate fluorescently labeled secondary anti- trol serum, was added to the culture medium at a concentration of 20 body (l-2 hr), and then postfixed for 10 set in an ethanol/acetic acid &ml. The IgG fractions were purified according to standard proce- mixture (95:5% v/v), and finally incubated with the second set of an- dures (Harlow and Lane, 1988). The sera were first precipitated with tibodies using the biotin-avidin detection system (Amersham). All an- 50%-saturated ammonium sulfate. The precipitate was then dissolved tibodies were appropriately diluted in blocking solution. Hybridoma and passed over a Protein A-Sepharose column (Pharmacia). The eluate supematant was used after adding 0.3% Triton X- 100. Fluorescence was from this column was again precipitated with ammonium sulfate, and visualized with a Zeiss epifluorescence microscope. the precipitate was dissolved and dialyzed extensively against HBSS. For whole-mount immunostaining, embryos of 6 d incubation were For each experiment, at least three independent trials were performed fixed in 4% formaldehyde/HBSS and incubated overnight in TBS (pH on different days. 7.6) containing 1 mM Ca2+, 0.3% Triton X-100, 1% bovine serum albumin, and 0.02% peroxide. The same solution without peroxide was then used for washes and antibody incubations, each lasting several Results hours to overnight. As the first antibody, NCD-2 was used. A bioti- N-cadherin expression in the spinal cord nylated secondary antibody was detected by streptavidin-biotinylated Figure IB shows the N-cadherin expressionpattern in a cross horseradish peroxidase (Amersham). The diaminobenzidine reaction product was visualized under a Zeiss light microscope. section of the E8 spinal cord. N-cadherin expressionis prom- DiI experiment. For tract tracing in the spinal cord with DiI (Honig inent in the dorsal funiculus of the , the floor plate and Hume, 1986) chick embryos of 3 d incubation were transferred (Shiga and Oppenheim, 1991) the periventricular region, and with the content of the egg into a Petri dish and incubated at 37°C in the radial . Outside the spinal cord, N-cadherin expression humid air following the procedure by Auerbach et al. (1974). The em- bryos were allowed to develop in vitro for 3 d until they had grown large is seenin the dorsal root ganglia and the dorsal root (Hatta et enough for a small crystal of DiI (Molecular Probes) to be inserted at al., 1987; Inuzuka et al., 1991b). the cervical level into the dorsal portion of the spinal cord using fine A comparison with the expressionpattern of pins. After 2 additional days of incubation, the embryos were fixed in the same section (Fig. 1A) reveals that only the lateral but not 4% formaldehyde/HBSS and processed for immunohistochemistry as the medial part of the dorsal funiculus is N-cadherin positive. described above. Cross sections ofthe spinal cord were obtained through the area of implantation and at successively more caudal levels. Within Figure 1, C and D, showsat a higher magnification that some 5 min after mounting cryostat sections on slide glasses, DiI was visu- fasciclesof the dorsal root expressN-cadherin while others do alized and photographed under fluorescent illumination using a Zeiss not. In general,the N-cadherin-positive fasciclesenter the spinal microscope. Waiting for longer than about 5 min resulted in increased cord more ventrally and merge with the N-cadherin-positive diffusion of DiI in the dried sections. As a control, consecutive sections region of the dorsal funiculus. In contrast, the N-cadherin-neg- were placed on slide glasses but immediately mounted in TBS without drying to prevent diffusion of DiI. Control results confirmed that there ative fasciclesenter the spinal cord more dorsally and merge was little DiI diffusion in the dried sections before photography (data with the N-cadherin-negative region either directly or after tra- not shown). Dried sections were then processed for double-label im- versing the N-cadherin-positive region of the dorsal funiculus munohistochemistry using anti-neurofilament and NCD-2 antibodies (Fig. lE,F). Similar results were obtained from E6 and El 1 as described above. Except for the implanted crystal of DiI, little or no DiI remained in the sections after immunostaining. The same sections embryos. that had been photographed earlier were analyzed again under epiflu- orescence illumination to obtain DiI, NCD-2, and neurofilament data Dil labeling of spinal cord from the same section. To examine whether the N-cadherin-positive and -negative cDNA transfection of neuroblastoma cells. Neuroblastoma cells of the Neuro 2a strain, which do not express any cadherin (Matsunaga et al., regionsin the dorsal funiculus representany particular structures 1988a), were grown in a I:1 mixture of Dulbecco’s modified Eagle’s of the spinal cord, we analyzed the relationship between the medium and Ham’s FI 2 medium supplemented with 10% fetal bovine patterns of N-cadherin expressionand DiI tract tracing (Honig The Journal of Neuroscience, September 1992, 12(9) 3527

Figure I. Double-stainingfor neuro- filament(A. C, E) and N-cadherin(B. D, F) in ihe thomcicspinal cord of ‘E8 (stage34) chicken.Note that only the lateralpart but not the medialpart of the dorsalfuniculus expresses N-cad- herin (A, B). The arrowsin C and D point to a dorsalroot fascicle,which doesnot expressN-cadherin. The ar- rowsin E andF pointto anN-cadherin- negativedorsal root fasciclewhich runs throughthe lateralregion of the dorsal funiculuswhich is N-cadherin positive. DRG, dorsalroot ;DR, dorsal root; DF, dorsalfuniculus. Scale bars: B, 100pm for A and B, F, 50 gm for C-F.

and Hume, 1986) of the sensorytract. DiI was implanted in the ganglia also contain DiI. These resultswere confirmed in other dorsal funiculus at a cervical level of E6 spinal cord, and the embryos and indicate that the medial part of the dorsal funiculus embryos were incubated for another 2 d, sectioned, and stained contains longer distance projections than the lateral part. for neurofilament and N-cadherin (Fig. 2). As shown in Fig. 2A-C, the entire dorsal funiculus is labeled with DiI at a level N-cadherin expressionin the hindbrain 0.5-l mm caudal to the DiI implant. In contrast, DiI labeling Figure 3 shows a longitudinal section through the pons and is absent from the N-cadherin-positive region at a level about medulla of an E8.5 embryo. Staining for N-cadherin (Fig. 3B) 3 mm caudal to the implant (Fig. 2D-fl. At this level, DiI is is seen only in a subset of the neurite fascicles that express detected only in the N-cadherin-negative region of the dorsal neurofilament (Fig. 3A). The N-cadherin-positive fibers extend funiculus. In both cases,parts of the dorsal roots and the sensory in a continuous band from the entry of the trigeminal nerve at 3525 Redies et al. l Cadherin Expression in Chicken Nervous System

Figure 2. Triple-staining of E8 chick- en spinal cord for neurofilament (A, D), for N-cadherin (B, E), and with DiI for tract tracing (C, F). DiI crystals were implanted into the cervical spinal cord. The section shown in A-C is 0.5-l mm caudal to the DiI implant, and that shown in SF is about 3 mm caudal to the DiI implant. The asterisks in D-F mark the lateral, N-cadherin-positive region of the dorsal funiculus, which shows no DiI label at this level. At the level closer to the DiI implant (A-C), the corresponding region contains DiI. Note that. at both levels, DiI label is present in the medial part of the dorsal funiculus. These sections are from an embryo incubated for 3 d in the egg and for another 5 d in a dish. Scale bars, 50 pm.

the rostra1 end of the pons to the caudal medulla oblongata. At glossopharyngeal,and vagus nerves contain nerve fasciclesex- a more caudal level, this N-cadherin-positive fiber tract extends pressingN-cadherin, whereasin the fascialand cochlea-acoustic into the lateral part of the dorsal funiculus of the spinal cord nerves, only glial elementsare N-cadherin positive. (data not shown). In Figure 4A, the samefiber tract can be seen In the trigeminal nerve, N-cadherin-positive fasciclesare found from a lateral viewpoint in a whole-mount preparation stained in the sensoryportion but not in the motor portion (Fig. 4B,C). for N-cadherin. Figures 3B and 4A show that the trigeminal, Furthermore, not all neurites in the sensory portion express The Journal of Neuroscience. September 1992, C?(9) 3529

- ,v

Figure 3. Double-staining of an E8.5 (stage 35) chicken hindbrain for neurofilament (A) and N-cadherin (Z?), and staining for R-cadhetin (c) in an adjacent section. These sections cut transversely through the pons and obliquely through the medulla oblongata (lower part of the sections). Arrows indicate R-cadherin-positive vagus motor fibers. V, root of trigeminal nerve; VZZ, VIII, roots of the facial and cochlea-acoustic nerves; ZX- A’, rootlets of the glossopharyngeal and vagus nerves; r, rostral; m, medial; c, causal, Z, lateral. Scale bar, 200 pm. 3530 Redies et al. l Cadherin Expression in Chicken Nervous System

d C + V

Figure 4. A, Lateral view of N-cadherin whole-mount staining of E6 (stage 29) chicken hindbrain. The arrowheads point to the ventral margin of the descending trigeminal tract. R V, root of the trigeminal nerve; R VIII, root of the cochlea-acoustic nerve; d, dorsal; c, caudal; v, ventral; r, rostral. B and C, Double-staining of an E8 (stage 34) chicken for neurofilament (B) and N-cadherin (C) in a cross section through the lateral pons at the level of the trigeminal nerve entry. The asterisk marks the region of the sensory trigeminal neurites that do not express N-cadherin. Arrows point to the minor (motor) portion of the trigeminal nerve, which shows no N-cadherin immunoreactivity. Scale bars: A, 200 mm; B and C, 50 urn.

N-cadherin. At the entry zone of the trigeminal nerve, N-cad- structures reach the brain surface after traversing N-cadherin- herin staining is only observed in a comma-shapedzone di- negative tissue (Fig. 3C). A close inspection reveals that most rectly below the surfaceof the pons. The neurofilament-positive of the fasciclesin the vagus nerve rootlets are either R-cadherin fasciclesthat penetrate the pons more deeply do not express positive or N-cadherin positive but rarely both (Fig. 5B-E). N-cadherin. In the deeperregions, fine fibers expressingN-cad- herin but not are found. These fibers likely Outgrowth of sensory axons on N-cadherin substrate representthe processesof radial glia. To test whether N-cadherin in sensoryaxons is actively involved in their migration, E6 sensoryganglia were explanted on N-cad- R-cadherin expression herin-transfected and control cell lines (Fig. 6). On untrans- R-cadherin is expressedin various regions of the spinal cord, fected cells that do not expressN-cadherin (Fig. 64, neurite most strongly in the midline region. At the embryonic stages outgrowth is strongly fasciculated. On N-cadherin-transfected studied here, there is no R-cadherin expressionin the sensory cells (Fig. 6B), a strong defasciculation is observed that can be and motor fibers of the spinal and cranial nerves (Inuzuka et blocked by purified anti-N-cadherin IgG (Fig. 6C). In those al., 1991 b). However, we found one exception: R-cadherin is areaswhere defasciculationoccurs, axons grow in a wavy, curved expressedin the motor nuclei of the vagus and glossopharyngeal fashion (Fig. 6B,D). In its form and periodicity, this axonal nerves, which are located close to the midline (Fig. 5A), and in growth pattern resemblesthe cell-cell boundary pattern of the their processesthat extend to the lateral surfaceof the brainstem N-cadherin-transfected cells (Fig. 60, G). Figure 6E showsmore (Fig. 3C). Here, they exit the brain by joining the N-cadherin- clearly that the axons originating from sensoryneurons follow positive (sensory)fibers that are entering the brain (Fig. 5&C). the cell-cell boundaries. In the presenceof anti-N-cadherin IgG, The sensoryfibers do not extend beyond the N-cadherin-pos- the axons grow much straighter (Fig. 60. For comparison, the itive zone of the descendingtrigeminal tract (Fig. 3B), whereas N-cadherin expressionpattern of the samecells is given in Fig- the R-cadherin-positive (motor) fibers originating from midline ure 6G. The Journal of Neuroscience, September 1992, 72(9) 3531

Figure 5. N- and R-cadherin expression in the vagus motor nuclei and nerve. A, R-cadherin staining of El 1 (stage 37) medulla. This is a cross section through the midline with the ventricular surface facing up. Note the positive immunoreactivity in the motor nuclei of the vagus nerve. B- E, Double-staining for N-cadherin (B, D) and R-cadherin (C, E) of the vagus nerve at its entry point into the medulla. Band Care from a transverse section of the medulla from an E8.5 (stage 35) chick embryo. D and E are from a cross section from an El 1 (stage 37) chick embryo. Arrowspoint to fascicles that express R-cadherin but not N-cadherin. Arrowheadspoint to fascicles that express N-cadherin but not R-cadherin. Scale bars: A, 50 pm; B-E, 100 pm.

are similarly distributed in a restricted, developmentally regu- Discussion lated way. N-cadherin and R-cadherin expressionare topographically restricted N-cadherin is d@erentialiy expressedin two types of dorsal In this study, we show that, in the CNS of E6-El1 chicken, root fascicles N-cadherin (Hatta et al., 1988) is expressedin a restricted fash- N-cadherin is expressedonly in a subsetof fasciclesof the spinal ion in a specific fiber tract, the general somatic sensorysystem dorsal root. In the dorsal funiculus into which most of the dorsal (Figs. 1, 3-5). At the sametime, another cadherin, R-cadherin root fibers extend, only the lateral part is N-cadherin positive. (Inuzuka et al., 1991a,b), is expressedby a different system, the Figure 1 demonstratesthat these two fiber systemsare appro- visceral motor component of the vagus and glossopharyngeal priately connected so that dorsal root fasciclesthat are positive nerves (Figs. 3-5). These patterns of expressionare observed at for N-cadherin join the lateral part of the dorsal funiculus while a time in development during which axons are actively growing. the negative neuritesjoin the medial part. Little or very low levels of N- and R-cadherin are present in The results from the DiI experiments suggestthat thesetwo the adult brain (Inuzuka et al., 1991a; C. Redies and M. Tak- portions of the dorsal funiculus are not equivalent but contain eichi, unpublishedobservations). It is conceivable that the other at least two different fiber systems. Whereas the medial part cadherinsrecently found in brain tissue(Napolitano et al., 1991; contains fibersextending rostrally over relatively largedistances, Ranscht and Dours-Zimmermann, 1991; Suzuki et al., 1991) the lateral, N-cadherin-positive region does not (Fig. 2). This 3532 Rediee et al. l Cadherin Expression in Chicken Nervous System

Figure 6. Axonal outgrowthfrom E6 (stage28-29) chickenexplants on N-cadherin-transfectedand control neuroblastomacells. Neurites are visualizedby immunofluorescentstaining with antibodiesagainst neurofilaments. In D-F, the underlyingmonolayer is visualized by superimposing the phase-contrastimages onto the neurofilamentimages. A, Outgrowthon the N-cadherin-negativecell line 2a-01in the presenceof 20 Mgof control IgG. B, Outgrowth on cell line Za-Ncadl,which expressesN-cadherin, in the presenceof 20 pg of control IgG. C, Outgrowthon cell line Za-Ncad1 in the presenceof 20 pg of anti-N-cadherinIgG. Note that neuritesare defasciculatedin B but not in A and C. D, Highermagnification of the axonaloutgrowth pattern on line Za-Ncad1. E andF, Singlesensory growing on line Za-Ncad1 without (E) and with Q the addition of 20 mg of anti-N-cadherinIgG to the culture medium.The urrows point to the bodiesof the bipolarsensory neurons. Note that the neuritesin E growalong the cell-cellboundaries whereas the neuritesin F growstraight. G, Line Za-Ncad1 stainedfor N-cadherin.High amounts of N-cadherin are expressedalong the cell-cellboundaries. Scale bars: C, 200 pm for A-C, E, 50 Mmfor D-G. finding confirms studiesin the chicken demonstrating that the brain in the respective ganglia. Inside the brain, these fibers medial part of the dorsal funiculus contains the longitudinal together form the N-cadherin-positive descendingtrigeminal long-distanceprojections running up and down the spinal cord tract (Figs. 3B, 4.4) which projects caudally and carriesgeneral while the lateral part contains mostly short-distanceprojections somatic sensoryinformation (Ramon y Cajal, 1911; Bok, 1915). (Davis et al., 1989). The N-cadherin-positive fibers likely cor- We found that the N-cadherin staining of this tract forms a respond to the “external fascicle” of the dorsal root described continuous transition to the lateral part of the dorsal funiculus by Ramon y Cajal and his contemporaries (Ramon y Cajal, of the spinal cord. The fact that both tracts expressN-cadherin 1911). This fascicle contains fine fibers that terminate, after a and their transition suggeststhat they share common devel- relatively short longitudinal run, in the dorsal horn of the spinal opmental features. cord gray matter. The N-cadherin-negative fibers may corre- The N-cadherin-negative fibersof the major (sensory)portion spond to the “internal fascicle” (Ramon y Cajal, 191l), which of the trigeminal nerve (Fig. 4C,D) passthrough the superficially contains large-diameter fibers giving rise to long-distance pro- located descendingtrigeminal tract. The minor portion of the jections after bifurcating into ascendingand descendingbranch- trigeminal nerve, a separatefiber bundle located more ventrally, es. Thus, the N-cadherin-positive and -negative regions of the doesnot expressN-cadherin; it carriesefferent (motor) neurites dorsal funiculus constitute distinct domains in axon migration. (Ramon y Cajal, 1911; Bok, 1915). Like the dorsal root of the A schematic summary of the immunostaining results and the spinal cord, the trigeminal nerve thus contains different fiber neuroanatomical correlates is given in Figure 7A. types of which the N-cadherin+expressingfibers form a unique subsetwith a separateprojection inside the CNS. N-cadherin is expressedin somatic sensoryfibers of the cranial nerves Topographical coincidence of N-cadherin expressionand In the brainstem, N-cadherin is expressedin sensory fascicles aberrant retinal projections of the trigeminal, glossopharyngeal,and vagus nerves (Fig. 3). The topographical distribution of N-cadherin expressionin the The nerve cell bodies of these axons are located outside the chicken CNS closely coincides with the pathstaken by aberrant The Journal of Neuroscience, September 1992, f.?(g) 3533

A spinal cord 6 medulla

d r

C ii V --- @II N’ -..- gj R’ E 13 N-IR-

Figure 7. Schematic three-dimensional representation of the immunostaining results and their nemoanatomical correlates in the spinal cord (A) and medulla (B) of the chicken. Shadings indicate immunohistochemical staining results. Broken lines indicate the expression of N- or R-cadherin by hypothetical individual neurites, assuming that neurites in a fiber tract express the molecule detected in this tract immunohistochemically. DRG, ; DR, dorsal root; DF, dorsal funiculus; d, dorsal; r, rostral; V, ventral; c, caudal; N+, N-cadherin positive; R+, R-cadherin oositive: N-/R-. negative for both N- and R-cadherin; ?, immunostaining pattern unknown; RX, root of vagus nerve; DV, descending trigeminal tract; NJ’, motor nucleus of the vagus nerve. projections from transplanted retinas induced in the frog (Con- sensory axons can use N-cadherin as a specific cell adhesion stantine-Paton and Capranica, 1976; Constantine-Paton, 1978). molecule in vivo. The segregationof N-cadherin-positive and In the brainstem, the aberrant projections follow the courseof -negative sensory fibers, found in the present study, thus may the descending trigeminal tract, and in the spinal cord, they depend at least partly on N-cadherin-mediated specificcell-cell typically grow in the dorsolateral, but not dorsomedial, white adhesion.For example, N-cadherin-positive sensoryaxons might matter tract (Constantine-Paton, 1978). In the chicken, both selectively migrate on preexisting axons or on glial cells that tracts expressN-cadherin (Figs. 1, 3, 4A), as do retinal axons expressthe samemolecule, resulting in the formation of N-cad- (Matsunaga et al., 1988a). If one assumesthat frog and chicken herin-positive and -negative fascicles in the sensory tract of are similar in their expressionof N-cadherin, thesefindings may the spinal cord. The same mechanism could operate for the indicate that N-cadherin servesto guide vertebrate retinal axons segregationof N-cadherin-positive and -negative sensoryfibers along the aberrant pathways by a homophilic adhesion mech- of the trigeminal nerve at their entry into the brain (Fig. 4B,C). anism. This mechanism may work in conjunction with other moleculeslike A5 (Takagi et al., 1987, 199l), whose expression A simple, cadherin-basedmodel for the segregationof motor is similarly restricted to the developing retinotectal system and and sensoryjibers of the vagus nerve the descendingtrigeminal tract ofthe frog (Fujisawaet al., 1989). It has been shown that, while N-cadherin and R-cadherin can mediate cell-cell aggregationvia their heterophilic binding, both N-cadherin as a guidance cuefor axon elongation prefer their own type (Inuzuka et al., 1991a). It is therefore N-cadherin provides a potent substratefor axon outgrowth from conceivable that N- and R-cadherin cause the segregationof retinal ganglion cells (Matsunaga et al., 1988; Drazba and Lem- two fiber types each expressingonly one of these cadherins. mon, 1990) and chick ciliary ganglion neurons(Tomaselli et al., Figure 7B illustrates this point for the sensoryand motor fibers 1988; Bixby and Zhang, 1990). Furthermore, Doherty et al. of the vagus nerve. R-cadherin, but not N-cadherin, is expressed (199 1) found a correlation between the length of rat cerebellar by the cell bodies of the visceral motor neurons located inside neurites and the level of N-cadherin expressionby the cells on the CNS closeto the midline. The R-cadherin-positive neurites which the neurites were growing. The restricted expressionpat- originating from thesenuclei traverse the brainstem and reach tern of N-cadherin in the developing CNS (Figs. 1, 3) suggests the superficially located region ofthe descendingtrigeminal tract that this molecule, like some membersof the immunoglobulin that expressesN-cadherin (Figs. 3, 5). Here, the R-cadherin- superfamily (seeintroductory remarks), may provide a cue for positive motor axons largely ignore the descendingsensory tract the directed growth of axons along preformed, molecularly de- and grow straight through this tract to exit the brain via the fined pathways. vagus nerve rootlets (Fig. 5) (Ramon y Cajal, 19 11; Bok, 19 15). Resultsfrom the present study support this hypothesis. The On the other hand, when the N-cadherin-positive sensoryfibers fact that, on N-cadherin-transfected cells, the sensory neurites of the vagus nerve enter the brain, they encounter the N-cad- defasciculateand grow along the cell-cell boundariesof the un- herin-positive descendingtrigeminal tract, which causesthem derlying monolayer (Fig. 6) implies that the axons more strongly to turn their direction along this tract and to segregatefrom the adhere to cell-cell boundaries than to themselves, perhapsbe- R-cadherin-positive motor fibers. causeN-cadherin is presentin higher amountsat the boundaries The weaker, heterophilic adhesion mechanismbetween N- than on the free surface of the transfected cells (Fig. 6G) and and R-cadherin (Inuzuka et al., 1991a) may mediate the rela- the sensoryaxons themselves.This finding strongly suggeststhat tively looseapposition of N-cadherin-positive sensoryfascicles 3534 Redies et al. * Cadherin Expression in Chicken Nervous System

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