cadherin expression in the retina and retinofugal pathways of the chicken embryo

Cadherin Expression in the Retinaand Retinofugal Pathways

of the Chicken Embryo

JENS-C.P. WOHRN,1 LUIS PUELLES,2 SHINICHI NAKAGAWA,3

MASATOSHI TAKEICHI,3 AND CHRISTOPH REDIES1,4*1Institute of Biology III, University of Freiburg, D-79104 Freiburg, Germany

2Department of Morphological Sciences, University of Murcia, Murcia 30100, Spain3Department of Biophysics, Faculty of Sciences, Kyoto University, Kyoto 606, Japan

4Institute of Anatomy, University Hospital Essen, D-45122 Essen, Germany

ABSTRACTThe expression of two calcium-dependent adhesion molecules of the cadherin superfamily

(cadherin-6B and cadherin-7) was mapped in the embryonic neural retina and retinofugalpathways of the chicken embryo and compared with the expression of R-cadherin, N-cadherin,and B-cadherin, studied previously. Whereas B-cadherin is only found in Muller glia, the otherfour cadherins are each expressed by specific subpopulations of retinal neurons. For example,different (but partly overlapping) populations of bipolar cells express R-cadherin, cadherin-6B, and cadherin-7. Cadherin-6B and cadherin-7 are also expressed by subsets of amacrinecells. In the inner plexiform layer, cadherin-6B and cadherin-7 immunoreactivities arerestricted to specific sublaminae associated with synapsin-I-positive nerve terminals. Inaddition, cadherin-6B and cadherin-7 are expressed by a subset of ganglion cells that projectto several retinorecipient nuclei forming part of the accessory optic system (e.g., nucleus of thebasal optic root and external pretectal nucleus). Together with their connecting fiber tracts,these nuclei also express cadherin-6B and cadherin-7 in their neurons and neuropile. Theexpression patterns of the two cadherins overlap but show distinct differences. Some othervisual nuclei express cadherin-7 but not cadherin-6B. The expression patterns differ fromthose previously described for N- and R-cadherin. Together, these results demonstrate thatcadherins could provide a system of adhesive cues that specify developing retinal circuits andother functional connections and subsystems in the embryonic chicken visual system.J. Comp. Neurol. 396:20–38, 1998. r 1998 Wiley-Liss, Inc.

Indexing terms: neural pathways; central nervous system; cell adhesion molecules; diencephalon;

accessory optic system

The vertebrate retina is composed of distinct sets ofneurons such as photoreceptor cells, bipolar cells, ganglioncells, and various types of interneurons. Their cell bodiesare arranged in three main layers (inner and outer nuclearlayers and ganglion cell layer). Between these layers,neurons are functionally connected to each other in theneuropile of the inner and outer plexiform layers. Each celltype shows a characteristic morphology, connectivity, andlocation in the retina (reviewed in Ramon y Cajal, 1911;Rodieck, 1973). Several cell types are also characterized bythe expression of specific molecules such as neurotransmit-ters (e.g., see Fletcher and Kalloniatis, 1996), their recep-tors (e.g., see Hering and Kroger, 1996), enzymes related totheir metabolism (e.g., see Millar et al., 1987), and interme-diate filament-associated proteins (e.g., see Drager et al.,1984).

The retina is connected to other structures in the centralnervous system (CNS) via the retinofugal projection, whichconsists of the optic nerve, optic chiasm, and optic tract.The fibers forming this projection originate in the ganglion

Grant sponsor: Land Baden-Wurttemberg Neurobiology Programme;Grant sponsor: Spanish Ministerio de Relaciones Exteriores and GermanAcademic Exchange Service (Acciones Integradas); Grant number: HA1996–0149; Grant sponsor: Human Frontier Science Programme; Grant number:GR-41–95; Grant sponsor: DGICYT Governmental Grant Agency, Madrid;Grant number: PB93–1137.

*Correspondence to: Christoph Redies, Institute of Anatomy, UniversityHospital Essen, Hufelandstrasse 55, D-45122 Essen, Germany.E-mail: [email protected]

Received 10 October 1997; Revised 5 February 1998; Accepted 10February 1998

THE JOURNAL OF COMPARATIVE NEUROLOGY 396:20–38 (1998)

r 1998 WILEY-LISS, INC.

cells of the retina. In birds, the largest projection field ofthe retinofugal fibers is the midbrain optic tectum (re-viewed in Rager, 1980; Mey and Thanos, 1992). However,optic nerve fibers also provide input to other targets, suchas several nuclei in the di- and mesencephalon (reviewedin Ehrlich and Mark, 1984; for comments on nomencla-ture, see Puelles et al., 1991).

In the present study, we describe the expression ofcadherins in the developing retina and retinofugal systemof the chicken. Cadherins are cell surface glycoproteinsthat regulate morphogenesis by mediating Ca21-depen-dent cell–cell adhesion (reviewed in Takeichi, 1995; Gum-biner, 1996). Cadherins were previously shown to beexpressed by particular brain nuclei and neural circuits inthe developing chicken and mouse CNS (reviewed inRedies, 1995, 1997; Redies and Takeichi, 1996). In thepresent study, the expression of cadherin-6B and cad-

herin-7 (Nakagawa and Takeichi, 1995) is mapped indetail. In addition, previously published results on theexpression of N-, R-, and B-cadherin in the visual system(Matsunaga et al., 1988b; Inuzuka et al., 1991; Redies etal., 1993; Redies and Takeichi, 1993; Murphy-Erdosh etal., 1994; Redies and Muller, 1994; Arndt and Redies, 1996;reviewed in Grunwald, 1996) are extended. Our resultsdemonstrate that, in the retina, the five cadherins areexpressed by particular types of neurons in a layer-specificfashion. Moreover, in the retinofugal pathway, expressionof cadherin-6B and -7 is largely restricted to a subset ofganglion cells and their processes. Some of the retinorecipi-ent nuclei of the mes- and diencephalon also expresscadherin-6B and -7. Together, these results demonstratethat cadherins are molecular markers for the functionalarchitecture of the visual system. The retinal pigmentepithelium was not the subject of the present study.

MATERIALS AND METHODS

Animals

Fertilized eggs from White Leghorn chicken (Gallusdomesticus) were purchased from a local farm. Eggs wereincubated at 65% humidity and 37°C in a forced-draftincubator (Ehret, Emmendingen, Germany). Embryos werekilled by decapitation, in accordance with the national andinstitutional guidelines on the use of animals in research.Embryos were staged according to Hamburger and Hamil-ton (1951). Embryos from the following stages were stud-ied: stage 11, stage 12, stage 14, stage 16, stages 18–21,stage 23, stage 26, stage 27, 6 days of incubation (E6; stage29), E7 (stage 31), E8 (stage 34), E9.5 (stage 35), E10(stage 36), E11 (stage 37), E14 (stage 40), E18 (stage 44),and E19 (stage 45).

Antibodies

The following specific antibodies were used: rat monoclo-nal antibody NCD-2 (Hatta and Takeichi, 1986) and rabbitpolyclonal antiserum (Hatta et al., 1988) raised againstchicken N-cadherin; mouse monoclonal antibody RCD-2raised against chicken R-cadherin (Redies et al., 1992;Arndt and Redies, 1996); mouse monoclonal antibodiesCC6B-1 and CC7–1 raised against chicken cadherin-6Band -7, respectively, and a rabbit polyclonal antiserumagainst chicken cadherin-6B (S. Nakagawa et al., unpub-lished observations); mouse monoclonal antibody 6D5(Angres et al., 1991) recognizing B-cadherin in chicken(Redies and Muller, 1994; kind gift of P. Hausen); rabbitpolyclonal antiserum raised against Xenopus synapsin I(Lu et al., 1996; kind gift of B. Lu); and rabbit polyclonalantiserum 1451 raised against chicken choline acetyltrans-ferase (ChAT; Millar et al., 1987; kind gift of M. Epstein).Mouse monoclonal antibody R5, which reacts with avimentin-associated protein, was used to demonstrateMuller cells in the retina (Drager et al., 1984; kind gift ofU. Drager). A polyclonal rabbit antiserum recognizing thecell adhesion molecule Ng-CAM/G4 in chicken (de la Rosaet al., 1990) was used to demonstrate fiber tracts (kind giftof E. de la Rosa). These antibodies were shown to reactspecifically with the respective molecules (for individualreferences, see above).

Appropriate secondary antibodies labeled with dichlo-rotriazinylamino fluorescein (DTAF), biotin, and the indo-carbocyanin dyes Cy2TM and Cy3TM were obtained fromDianova (Hamburg, Germany). Biotinylated secondary

Abbreviations

ApR perirotundic areaB-cad B-cadherinBOR basal optic rootcad 6B cadherin-6Bcad 7 cadherin-7ChAT choline acetyltransferaseCNS central nervous systemCO optic chiasmd dorsaldl nucleus of the oculomotor nerve, dorsolateral partDLL lateral part of the anterior dorsolateral complex of the

thalamusdm nucleus of the oculomotor nerve, dorsomedial partdsd dorsal supraoptic decussationdsv ventral supraoptic decussationE days of incubationEP external pretectal nucleusEW nucleus of Edinger-WestphalG4 Ng-CAM/G4 immunoreactivityGCL ganglion cell layerGlv lateral geniculate nucleus, ventral partGlv-c compact layer of lateral geniculate nucleus, ventral partGlv-p plexiform layer of lateral geniculate nucleus, ventral partGT griseum tectaleHb hindbrainHoe nuclear stain (Hoechst 33258)INL inner nuclear layerIPL inner plexiform layerITO interstitial nucleus of the optic tractl lateralLA nucleus lateralis anterior of ventral thalamusLS lateral suprachiasmatic nucleusnBOR nucleus of the basal optic root (ectomamillary nucleus)N-cad N-cadherinNE neuroepitheliumNFL nerve fiber layerNH optic nerve headOLM outer limiting membraneON optic nerveONL outer nuclear layerOPL outer plexiform layerOT optic tractPE pigment epitheliumPPC principal precommissural nucleusR nucleus rotundusR5 vimentin-associated antigenR-cad R-cadherinRet retinaSS superficial synencephalic nucleussyn synapsin Ithio thionine (Nissl) stainTO optic tectumv ventralVL ventrolateral nucleusvm nucleus of the oculomotor nerve, ventromedial part

CADHERIN EXPRESSION IN THE RETINA 21

antibody was detected with DTAF-labeled streptavidin(Dianova).

Immunohistochemistry

Immunostaining procedures were done as described indetail by Redies et al. (1992, 1993) and Arndt and Redies(1996). Heads from stage 15 to E8 chicken embryos andbrains and eyes from E9–E19 embryos were fixed for 1–3hours in 4% formaldehyde dissolved in Hanks’ balancedsalt solution supplemented with 1 mM Ca21 and 1 mMMg21 (HBSS) at 4°C. Subsequently, specimens were im-mersed in a graded series of sucrose solutions (12–18%sucrose), embedded in Tissue Tek O.C.T. medium (Sakura,Torrance, CA), frozen in liquid N2, and stored at 280°C.

Sections of 10–20 µm thickness were cut in a refriger-ated microtome, mounted on coated glass slides, and dried.Sections were postfixed for 10 minutes in 4% formaldehyde/HBSS at 4°C and washed in Tris-buffered saline (pH 7.6)containing 1 mM CaCl2 (TBS).

For fluorescence immunohistochemistry, sections wereincubated in a solution containing 5% skimmed milkpowder, 0.3% Triton X-100, and 0.04% sodium azide inTBS (‘‘blocking solution’’). Incubation with the first antibod-ies diluted in blocking solution was done for 1 hour orovernight, followed by incubation with the secondaryantibodies for 30–60 minutes. For double-label immuno-staining, previously published procedures were followed(Redies et al., 1992, 1993). To visualize cell nuclei, sectionswere counterstained with nuclear dye Hoechst 33258(Sigma, Deisenhofen, Germany). Sections were mountedeither in 90% glycerol/10% TBS or Vectashield medium(Vector Laboratories, Burlingame, CA). Fluorescence wasvisualized under an epifluorescence microscope (Zeiss Ax-ioplan, Oberkochen, Germany) and with a confocal laserscanning microscope (Leica, Heidelberg, Germany). Todisplay results from double-labeled sections simulta-neously (Fig. 5), confocal images were enhanced in con-trast, color-coded, and superimposed with a computer byusing Photoshop (Adobe, Mountain View, CA) and Free-hand (Multimedia, San Francisco, CA) image-processingprograms.

For peroxidase immunohistochemistry, a commerciallyavailable kit was used (Vectastain Elite ABC kit, VectorLaboratories), as described previously (Redies et al., 1997).To block unspecific binding and to dilute antibodies, 1.5%horse serum in TBS was used. Primary antibodies wereapplied for 1 hour at room temperature or overnight. Aftersequential incubation with appropriate biotinylated second-ary antibody and avidin-coupled peroxidase complex, sec-tions were incubated in substrate solution (0.7% 3–38diaminobenzidine tetrachloride, 0.5% nickel chloride, and0.1% peroxide in TBS). After reaction product had formed,sections were washed, dehydrated in an ascending ethanolseries, and embedded in Entellan (Merck, Darmstadt,Germany).

For neuroanatomical orientation, sections adjacent tothe immunostained sections were stained with thionineacetate (Sigma) for Nissl substance, as described previ-ously (Redies et al., 1993). All thionine stains shown in thefigures are brightfield photomicrographs.

Neuroanatomical structures were identified on atlasesof the adult pigeon brain (Karten and Hodos, 1967) and theadult chicken brain (Kuenzel and Masson, 1988). Theembryological analysis of Puelles et al. (1991) was alsofollowed. The nomenclature of these publications was

used. In addition, various other neuroanatomical publica-tions were consulted.

In situ hybridization

Sense and anti-sense digoxigenin-labeled RNAs weresynthesized in vitro by using commercially available kits(Stratagene, La Jolla, CA; Boehringer Mannheim, Mann-heim, Germany) and alkaline hydrolyzed into 300-bpfragments. The following templates containing full-lengthcDNAs (derivatives of pBluescript DNA vector) were used:pRcad (chicken R-cadherin; Inuzuka et al., 1991), Z10T6/BS(chicken N-cadherin; Hatta et al., 1988), nk-9 (chickencadherin-6B), and nk-2 (chicken cadherin-7; Nakagawaand Takeichi, 1995). For in situ hybridization, previouslypublished procedures were followed (Redies et al., 1993;Ganzler and Redies, 1995). Series of cryostat sectionsthrough the eyes were obtained from E5, E6, E7, E8, E10,E14, and E18 embryos. Digoxigenin-labeled probe hybrid-ized in situ was detected with anti-digoxigenin alkalinephosphatase-conjugated Fab fragments (Boehringer), fol-lowed by a reaction using X-phosphate and nitrobluetetrazolium salt as substrates. Sections embedded inEntellan (Merck) were viewed under a transmission lightmicroscope (Zeiss Axioplan). All in situ hybridization re-sults shown in the figures are brightfield photomicro-graphs.

RESULTS

Cadherin expression in the neural retina

General observations. Figures 1 and 2 show theexpression of five cadherins (N-cadherin, R-cadherin, cad-herin-6B, cadherin-7, and B-cadherin) in the central neu-ral retina of embryos of different ages (E5, E7, E9, E11,E14, and E18). For identification of the different retinallayers, a corresponding fiber stain (Figs. 1F,M,T, 2F,M,T)obtained with polyclonal antibodies against the cell adhe-sion molecule Ng-CAM/G4 (de la Rosa et al., 1990), and anuclear stain (Figs. 1G,N,U, 2G,N,U) are shown on theright side of each a series of panels. In general, eachcadherin is expressed in a characteristic spatiotemporalpattern. With the exception of N-cadherin (Matsunaga etal., 1988b), none of the other cadherins is expressed in theundifferentiated neural retina before E2.5 (stage 18). Theonset of expression varies between E2.5 and E9. In gen-eral, each cadherin is found in a subset of cell types withinparticular retinal layers, as described in detail in thefollowing sections. The structures expressing a givencadherin maintain expression at least until E18. Unlikethe other cadherins, N-cadherin is expressed uniformly inthe early neural retina (Fig. 1A; Matsunaga et al., 1988b;Inuzuka et al., 1991).

Cadherin-6B. Expression of cadherin-6B is first ob-served in single ganglion cells and their processes in thecentral retina at around E5 (arrow in Fig. 1C). As develop-ment proceeds, the number of cadherin-6B-positive gan-glion cells increases in the central retina (Fig. 1J) andexpression spreads peripherally. At E9, when the differentlayers of the retina have become more distinct, cad-herin-6B is found in a subset of cells in the ganglion celllayer (GCL; Fig. 1Q; large arrowhead in Fig. 3G) and inisolated cells of the inner nuclear layer (INL; Fig. 1Q). Forexample, cadherin-6B is expressed by small amacrine cellsin the inner nuclear layer (arrows in Fig. 3G) and by cellsmigrating through the inner plexiform layer (IPL) to the

22 J.-C.P. WOHRN ET AL.

ganglion cell layer between E9 and E10 (arrowheads inFigs. 1Q, 5J,K; small arrowheads in Fig. 3E–H; largearrowheads in Fig. 9B; Genis-Galvez et al., 1977). Compari-

son of cadherin-6B staining at E8–E9 with data reportedby Genis-Galvez et al. (1977) suggests that these cadherin-6B-positive cells are identical to the subset of multipodal

Fig. 1. Differential expression of cadherins in central regions of thechicken retina on day 5 of incubation (E5), E7, and E9. Immunofluores-cence results are shown. Sections were stained with antibodies againstN-cadherin (N-cad; A,H,O), R-cadherin (R-cad; B,I,P), cadherin-6B(cad 6B; C,J,Q), cadherin-7 (cad 7; D,K,R), B-cadherin (B-cad; E,L,S),Ng-CAM/G4 (G4; F,M,T), and with nuclear stain Hoechst 33258 (Hoe;

G,N,U). The different layers of the retina are indicated on the rightside of each series of panels. The arrows in C,D,J, and K point atganglion cells. The arrowheads in Q point at prospective invertedamacrine cells. The arrowhead in S points at the outer limitingmembrane (OLM). For other abbreviations, see list. Scale bar 550 µm.


Fig. 2. Differential expression of cadherins in central regions of thechicken retina on day 11 of incubation (E11), E14, and E18. Immuno-fluorescence results are shown. Sections were stained with antibodiesagainst N-cadherin (N-cad; A,H,O), R-cadherin (R-cad; B,I,P), cad-herin-6B (cad 6B; C,J,Q), cadherin-7 (cad 7; D,K,R), B-cadherin(B-cad; E,L,S), Ng-Cam/G4 (G4; F,M,T), and with nuclear stainHoechst 33258 (Hoe; G,N,U). The different layers of the retina areindicated on the right side of each series of panels. The small arrow inC points at a ganglion cell. The arrowheads in E,L, and S point at the

outer limiting membrane. The small arrowheads in H–K point atamacrine cells. The small arrows in J,K, and R point at bipolar cells.The large arrowhead in I points at the cell body of a Muller cell. Thelarge arrowheads in J and K point at small spots of immunofluores-cence in the outer limiting membrane. The large arrow in H points at ahorizontal cell. The small arrows in Q point at the outermost cadherin-6B-positive sublayer of the inner plexiform layer (IPL). The arrow-heads in Q point at a cadherin-6B-positive sublayer of the innerplexiform layer. For other abbreviations, see list. Scale bar 5 50 µm.

amacrine cells that adopt a transient bushy appearance atthe outer border of the inner plexiform layer (‘‘bushy’’ or‘‘short-stalked’’ amacrine cells; Prada et al., 1987). Thesecells are transiently found at a particular sublamina of theinner plexiform layer (Figs. 1Q, 3F,H, 5J–L, 9B) and later

develop into inverted amacrine cells (Genis-Galvez et al.,1977).

By E11, the inner plexiform layer displays two promi-nent narrow sublaminae of cadherin-6B immunoreactivity(Fig. 2C). These two sublaminae and the cadherin-6B-

Fig. 3. Cell type-specific expression of cadherins by retinal neu-rons. A–D: Displaced ganglion cells in the retina immunostained at 14days of incubation (E14) for cadherin-6B (cad 6B; A) and cadherin-7(cad 7; C). Corresponding nuclear stains (Hoe) are shown in B and D.Immunofluorescence results are shown. The displaced ganglion cells(arrows) are relatively large and contain a large cell nucleus. PairsA–B and C–D represent double-label results from the same section.The different layers of the retina are indicated on the right-hand side.E–H: Prospective displaced amacrine cells migrate through the innerplexiform layer (IPL) at E9. Immunofluorescence results are shown forR-cadherin (R-cad) in E, for cadherin-6B (cad 6B) in G, and forNg-CAM/G4 (G4) in H. A nuclear stain (Hoe) is shown in F. Pairs E–F

and G–H represent double-label results from the same section. Thesections are adjacent. The small arrowheads in E–H point at cellbodies of the migrating amacrine cells. The arrows in E point atR-cadherin-positive cell somata extending processes into the innerplexiform layer. The arrows in G point at amacrine cells in the innernuclear layer (INL). The large arrowhead in G points at a ganglioncell. I: Cell types expressing cadherin-6B (cad 6B) in the E18 retina.The large arowhead points at a displaced ganglion cell. The smallarrowheads points at bipolar cells. The arrows point at amacrine cellsin the inner nuclear layer (INL). For other abbreviations, see list.Scale bars 5 20 µm for A–D, E–H, I.


expressing ganglion cells and amacrine cells (large arrow-head and arrows, respectively, in Fig. 3I) retain theirimmunostaining at least until E18. In addition, a subset ofbipolar cells (arrowheads in Fig. 3I) and the outer plexi-form layer (OPL) begin to express cadherin-6B betweenE11 and E14. At E18, two additional sublaminae of faintcadherin-6B expression are seen in the inner plexiformlayer (Fig. 3I; arrows and arrowheads in Fig. 2Q).

In the ganglion cell layer, subsets of amacrine cells (datanot shown) and ganglion cells (large arrowhead in Fig. 3G)show cadherin-6B immunoreactivity. Ganglion cells areeither small, with a rectangular shape, or large, with around shape. Amacrine cells are small, round, and regu-larly spaced throughout the retina (Ramon y Cajal, 1911;Genis-Galvez et al., 1977).

Figure 3A shows a cadherin-6B-positive cell identified asa displaced ganglion cell; it is located in the inner nuclearlayer close to the border to the inner plexiform layer. Thistype of cell is relatively large (20–30 µm). Its position ismarked by the absence of small nuclei in the correspond-ing nuclear stain (arrow in Fig. 3B). Immunostaining ismore prominent on the cell surface and the axodendritictrunk. Faintly stained dendrites are sometimes seen ex-tending into the inner plexiform layer (data not shown). Aspreviously described by Ramon y Cajal (1911) and Fite etal. (1981), these processes contribute to the weakly stained,outermost sublamina of the inner plexiform layer (arrowsin Fig. 2Q). The displaced ganglion cells are widely spacedand show an irregular distribution. They are found morefrequently in peripheral and inferior regions of the retinaand are relatively sparse in the fovea and the red field (Fiteet al., 1981). A series of double-labeled sections through theentire E14 retina showed that all cells identified asdisplaced ganglion cells on the nuclear stain expresscadherin-6B.

In the inner nuclear layer, cadherin-6B-positive ama-crine cells are relatively rare (arrows in Fig. 3I). Thesecells are found at a similar level within the inner nuclearlayer as the ChAT-positive amacrine cells described byMillar et al. (1987). The ChAT-positive amacrine cells inthe inner nuclear layer are numerous and are eitherlocated adjacent to or remote from the inner plexiformlayer. Double staining with antibodies against cad-herin-6B and ChAT (Figs. 4A,B, 5B) demonstrates thatthere is no overlap between these two populations. Thesame result was obtained for the displaced amacrine cellsof the ganglion cell layer. Interestingly, ChAT and cad-herin-6B are expressed in the same two sublaminae of theinner plexiform layer (Figs. 4A,B, 5B). Double-labelingwith antibodies against the synaptic vesicle-associatedprotein synapsin I (Sudhof et al., 1989; Lu et al., 1996)shows a close association or coexpression of the twomolecules in both layers (arrowheads in Fig. 5H).

A subpopulation of bipolar cells also expresses cad-herin-6B in the outer part of the inner nuclear layer (arrowin Fig. 2J; small arrowheads in Fig. 3I). On some sections,their neurites can be followed from the inner plexiformlayer to the outer plexiform layer (data not shown). Thesecells resemble the small bipolar cells described by Ramon yCajal (1911). The processes often end in varicosities at theouter limiting membrane (‘‘Landolt’’ clubs, large arrow-heads in Fig. 2J). Individual cadherin-6B-expressing pro-cesses can sometimes be followed from the endfeet in theouter limiting membrane to individual bipolar cells in theinner nuclear layer (data not shown).

Figure 6C shows in situ hybridization results confirmingthe expression of cadherin-6B mRNA by the cell somata ofthe different cell types described above.

Cadherin-7. Low levels of cadherin-7 expression arefirst observed in the central retina at around stage 18

Fig. 4. Double-label immunostaining results for retina stainedwith antibodies against choline acetyltransferase (ChAT; A,C), cad-herin-6B (cad 6B; B), cadherin-7 (cad 7; D), vimentin-associatedantigen R5 (R5; E), and B-cadherin (B-cad; F) at 18 days of incubation.Immunofluorescence results are shown. Pairs A–B, C–D, and E–F

represent double-labeling results from the same section. Color-codedoverlays of pairs A–B and C–D are shown in Figure 5B,C. The differentlayers of the retina are indicated on the right side. Arrowheads in A–Dpoint at ChAT-positive cell bodies that do not express cadherin-6B orcadherin-7. For other abbreviations, see list. Scale bar 5 20 µm.


(E2.5) in the earliest differentiating ganglion cells (Pradaet al., 1991), in their neurites within the developing nervefiber layer, and in densely spaced radial processes span-ning the entire retina (data not shown). From stage 18 toE7, cadherin-7 expression by these structures increases(Fig. 1D,K) and spreads peripherally. Expression by theradial processes has greatly diminished by E11. Stainingof the ganglion cells and their processes diminishes fromE9 to E11 but persists at low levels at least until E18 (largearrow in Fig. 7A). At E14, cadherin-7 is expressed by abouthalf of the cells identified as displaced ganglion cells by thenuclear stain (Fig. 3C,D).

As the expression by the dense radial processes dimin-ishes, several other cadherin-7-positive cell types can bediscerned (Fig. 2D,K,R), as described in the followingparagraphs. A comparison with the nuclear stains of thesame sections demonstrates that only subsets of thedifferent retinal cell types express cadherin-7 (data notshown).

In the ganglion cell layer, displaced amacrine cellsexpressing cadherin-7 are rare, very faintly stained, andirregularly distributed (data not shown). In the innernuclear layer, cadherin-7-positive amacrine cells are nu-merous, clearly stained, and found at different positionswithin this layer (large arrowheads in Fig. 7). Doublestaining with antibodies against ChAT (Figs. 4C,D, 5C)shows no overlap between the cadherin-7-positive andChAT-positive populations of amacrine cells.

Of the bipolar cells in the inner nuclear layer, only asubset expresses cadherin-7 (small arrow in Fig. 2J; smallarrowheads in Fig. 7A). The processes of these cells arealso immunoreactive and project to the inner and outerplexiform layers (small arrows in Fig. 7A). Some of theprocesses reach the outer limiting membrane in a waysimilar to that described for the Landolt clubs (largearrowheads in Fig. 2K; arrows in Fig. 7B; Ramon y Cajal,1911; Quesada and Genis-Galvez, 1985). The cadherin-7-positive bipolar cells form a relatively broad band in theouter one-third of the inner nuclear layer, as confirmed byin situ hybridization (arrows in Fig. 6D).

Double labeling with antibodies against cadherin-6Band -7 shows partial overlap between the populations ofpositive cells. For example, some (but not all) ganglioncells, displaced ganglion cells, displaced amacrine cells,and bipolar cells express both molecules (Fig. 5A).

The inner plexiform layer shows widespread cadherin-7expression, but some sublaminae display higher expres-sion levels than others (Figs. 2D,K,R, 3C, 4D, 7). Thesublaminae positive for ChAT and cadherin-6B are asubset of the cadherin-7-positive sublaminae (Figs. 4A–D,5B,C). Double labeling with antibodies against synapsin Ishows that most, if not all, cadherin-7 immunoreactivity isassociated with synapsin I staining. However, not allsynapsin-I-positive sublaminae express cadherin-7 (Fig. 5I).

B-Cadherin. B-cadherin is ubiquitously expressed atearly developmental stages (E3) in peripheral regions ofthe neural retina (Redies and Muller, 1994). This expres-sion subsequently disappears. The retinal pigment epithe-lium is also B-cadherin positive (Fig. 1E,L,S; Murphy-Erdosh et al., 1994; Redies and Muller, 1994).

Expression of B-cadherin in the neural retina reappearsat E6.5–E7 in central regions and subsequently spreadsperipherally. This expression has a filamentous appear-

ance and radially spans the entire thickness of the neuralretina, from the outer limiting membrane to the nervefiber layer (Fig. 1S). From E9 to at least E18, staining isespecially pronounced and uniform in the outer limitingmembrane (OLM; arrowheads in Figs. 1S, 2E,L,S). At E11,filamentous staining is prominent in the nerve fiber layer,the ganglion cell layer, the inner nuclear layer, and theouter plexiform layer (Fig. 2E). Because the temporal andspatial profile of B-cadherin expression resembles that ofthe Muller glia population (Prada et al., 1989), E18 retinalsections were double stained with antibodies against B-cadherin and the intermediate filament-associated anti-gen R5, a marker for Muller glial cells (Fig. 4E,F; Drager etal., 1984). Results of confocal microscopy show extensivecoexpression of the two antigens in the processes of theMuller cells (Fig. 5D). The R5 antigen (but not B-cadherin)is also expressed in the Muller cell bodies. With theexception of the pigment epithelium, no other structures ofthe chicken visual system was found to express B-cadherinin the present study.

N- and R-cadherin. The expression of N-cadherin andR-cadherin in the chicken retina was described previously(Matsunaga et al., 1988b; Inuzuka et al., 1991). Like thecadherins described above, N-cadherin and R-cadherin aredifferentially expressed in the nuclear and plexiform lay-ers, with each cadherin labeling different subsets of cellsand sublayers (Figs. 1, 2). For example, at E14, onlyN-cadherin is expressed by photoreceptor cells of the outernuclear layer (ONL; Figs. 2H, 6A) and by horizontal cells ofthe inner nuclear layer (large arrow in Figs. 2H, 6A).R-cadherin expression begins at around E9 and is seenimmediately adjacent to the inner plexiform layer (Figs.1P, 2B,I,P). In the inner nuclear layer, expression ofR-cadherin is slightly more pronounced in its middleregion containing the perikarya of Muller cells and aninner group of bipolar cells (Figs. 2I, 6B; arrows in Fig. 3E),whereas the perikarya of the bipolar cells located close tothe outer plexiform layer express cadherin-6B (Figs. 2J,6C; arrowheads in Fig. 3I). Both sublaminae containcadherin-7-positive bipolar cells (Figs. 2K, 6D). The fourcadherins are also differentially expressed by subsets ofamacrine cells (small arrowheads in Figs. 2H–K, 6A–D).The in situ hybridization results for E14 (Fig. 6) and forother stages (E5, E6, E7, E8, E10, and E18; data notshown) confirm the immunostaining results illustrated inFigures 1 and 2.

There is little association among N-cadherin, R-cad-herin and synapsin I immunostaining (Fig. 5F,G). Rather,the expression patterns of these molecules are largelycomplementary. Interestingly, an outer sublamina of theinner plexiform layer is densely immunoreactive for R-cadherin at E8–E9, when prospective inverted amacrinecells form a transient intraplexiform cell line (Fig. 1P,Q;arrowheads in Figs. 3E,F, 5J–L, 9A,B,D; Genis-Galvez etal., 1977).

Optic pathways

Only a subset of retinal ganglion cells expresses cad-herin-6B and cadherin-7. In the optic pathway (opticnerve, chiasm, and tract), which is formed by the processesof the ganglion cells, only a relatively small subset of fibersexpresses these two molecules (Figs. 8C,D, 9B,C). Incontrast, most (if not all) optic fibers express N-cadherin


(Figs. 8A, 9E,F; Matsunaga et al., 1988a), whereas R-cadherin (Figs. 8B, 9A; Redies and Takeichi, 1993) andB-cadherin (Fig. 2E,L,S) are absent from ganglion cells or

their processes. The R-cadherin immunoreactivity seen inthe optic nerve (Fig. 8B) is associated with astroglia(Redies and Takeichi, 1993).

Figure 5


Cadherin-7-positive optic fibers are first observed in theretinal nerve fiber layer at E2.5. Cadherin-6B-expressingneurites appear later (around E5). For cadherin-7, thestaining intensity of the optic pathway increases andreaches its maximum at around E8–E9. It then decreasesagain until E14. Cadherin-6B expression initially alsoincreases but then remains at a relatively high level fromE8 to E18 (data not shown). For a closer analysis of the

distribution of cadherin-positive fibers in the optic path-way, we focused on the E8–E9 expression patterns. At thisstage of development, many optic fibers have alreadyreached the optic tectum (De Long and Coulombre, 1965;Goldberg, 1974; Crossland et al., 1975; Mey and Thanos,1992).

The fiber populations expressing cadherin-6B and cad-herin-7 grow largely within the outer half of the retinalnerve fiber layer (adjacent to the ganglion cell bodies; Figs.8C,D, 9B,C). At E8, these neurites can be seen to originatefrom ganglion cells in the entire retina. As the neuritesapproach the optic nerve head, they fan out into the opticnerve (Figs. 8C,D, 9B,C), apparently mixing with otherfibers stained with the N-cadherin antibody. In the opticnerve head, the cadherin-6B-positive fibers run in closeapposition to the early optic nerve glia, represented byimmature type I-like astrocytes, which can be visualizedby R-cadherin immunostaining (arrows in Figs. 5J–L,9A,D; Redies and Takeichi, 1993). These astrocytes alsoexpress cadherin-7 (arrows in Figs. 8D, 9C, 10D). Neuroepi-thelial cells lying at the outer rim of the optic nerve alsostrongly express R-cadherin and cadherin-7 (NE in Figs.8B,D, 10B,D).

In the optic chiasm, the neurites expressing cadherin-6Band cadherin-7 are segregated from fibers that do notexpress the two cadherins, and they form thick fiberbundles (arrowheads in Fig. 10C,D). Farther into theircourse in the optic tract, the neurites expressing cad-herin-6B and cadherin-7 assume a deep position in directapposition to the di- and mesencephalic gray matter(arrowheads in Fig. 11A). Here, the less numerous cad-herin-7-positive fibers are among the deepest fibers. In thebasal optic root (BOR in Fig. 11D,E), some cadherin-6B-positive fibers run superficially. In the E11 optic chiasmand tract, the cadherin-7-expressing areas coincide withthe cadherin-6B-positive ones but the area occupied by thelatter is slightly more extensive (Figs. 10C,D, 11A,B).

Fig. 6. Cadherin mRNA expression in the retina at 14 days ofincubation. Sections were hybridized with anti-sense probe for N-cadherin (N-cad; A), R-cadherin (R-cad; B), cadherin-6B (cad 6B; C),and cadherin-7 (cad 7; D). A neighboring section stained with thionine(thio) for Nissl substance is shown in E. Cadherin transcript is

indicated by the dark reaction product. Arrowheads in A–D point atamacrine cells. Small arrows in B–D point at bipolar cells. The largearrow in A points at a horizontal cell. The different layers of the retinaare indicated on the right-hand side. For other abbreviations, see list.Scale bar 5 50 µm.

Fig. 5. Color-coded overlays of double-staining results for cadher-ins and various other markers. Results shown are from the retina at18 days of incubation (E18; A–D), the E11 ventromedial oculomotornucleus (E), the E14 inner plexiform layer (F–I), and the E8.5 retina atthe transition to the optic nerve head (J–L). A represents results for asection stained with antibodies against cadherin-6B (red, cad6b) andcadherin-7 (green, cad7) obtained with confocal laser scanning micros-copy. B and C represent overlays of the data shown in Figure 4A,B and4C,D, respectively. Cadherin-6B (B) and cadherin-7 (C) immunoreac-tivity is shown in red. ChAT immunoreactivity is shown in green. Drepresents results for a section stained with antibodies against B-cadherin(red, B-cad) and vimentin-associated antigen R5 (green) obtained withconfocal laser scanning microscopy. Many processes coexpress R5 andB-cadherin (yellow). E represents an overlay of the data shown in Figure13D,E. Cadherin-6B immunoreactivity is shown in red. Cadherin-7 immu-noreactivity is shown in green. Arrowheads point at neurons expressingboth cadherin-6B and cadherin-7 (yellow). Arrows point at neurons exclu-sively expressing cadherin-6B. F–I show double-label confocal results fordifferent cadherins (red; F: N-cad, N-cadherin; G: R-cad, R-cadherin;H: cad6b, cadherin-6B; I: cad7, cadherin-7) and for synapsin I (green, syn).The arrowheads in H and I point at two sublaminae of the inner plexiformlayer strongly coexpressing synapsin I (H) and cadherin-6B and cadherin-7(I). J–L shows sections from an area similar to that boxed in Figure 8B andshown at a higher magnification in Figure 9A–F. J represents resultsobtained with antibodies against cadherin-6B (red, cad6b) and nuclearstain Hoechst 33258 (green, Hoe). K represents results obtained withantibodies against cadherin-6B (red, cad6b) and R-cadherin (green, R-cad).L represents results obtained with antibodies against R-cadherin (red,R-cad) and nuclear stain Hoechst 33258 (green, Hoe). Arrowheads point atprospective inverted amacrine cells. Arrows point at R-cadherin-positiveglial cell columns of the optic nerve. For other abbreviations, see list.Scale bars 5 20 µm in A–E, 10 µm in F–I, 100 µm in J–L.


Retinorecipient nuclei and accessoryoptic system

The superficial layers of the tectum, the major target ofthe optic fibers, differentially express N-cadherin, R-cadherin (Redies et al., 1993), cadherin-6B, and cadherin-7(J.-C. P. Wohrn and C. Redies, unpublished observations).A minor portion of the optic fibers projects to various mes-and diencephalic nuclei (Ehrlich and Mark, 1984; Britto,1989; Puelles et al., 1991; Shimizu et al., 1994). Thesenuclei are of special interest because most of them arecharacterized by the expression of cadherin-6B, cad-herin-7, or both. It should be noted that, in principle, theimmunoreactivity observed in these nuclei can be due toexpression by intrinsic cells, by optic nerve fiber terminals,or by other afferents expressing these cadherins. Expres-sion data for cell somata were confirmed by in situhybridization controls.

Cadherin-6B is strongly expressed by cells and neuro-pile of the nucleus lateralis anterior (LA in Fig. 11A), witha stronger signal in its ventral half. The ventral geniculatenucleus shows cadherin-6B-immunoreactive cells in itscompact deep lamina (Glv-c in Fig. 11A,D), with a strongersignal in its caudal part. Invasion of its superficial plexi-

form lamina by immunoreactive fibers may affect only thesuperficial part (Fig. 11D). Cadherin-6B is also expressedby the external pretectal nucleus (EP in Fig. 11D; alsoknown as nucleus lentiformis mesencephali, pars magno-cellularis, and nucleus of the basal optic root, pars latera-lis), the nucleus of the basal optic root (nBOR in Fig. 12),and the area pretectalis (data not shown). Cadherin-7 isalso expressed by cells and neuropile in these nuclei (Figs.11B,E, 12C,D). In addition, the griseum tectale (GT inFigs. 11D,E, 12B,C), the superficial synencephalic nucleusof Rendahl (SS in Figs. 11D,E, 12B,C), the lateral part ofthe anterior dorsolateral complex of the thalamus (DLL inFig. 11E,F), the perirotundic area (ApR in Fig. 11E,F), andthe ventrolateral nucleus (VL in Fig. 11B,C) expresscadherin-7. All of them are retinorecipient structures(Ehrlich and Mark, 1984; Martınez et al., 1991; Shimizu etal., 1994).

In general, the nuclei that coexpress cadherin-6B andcadherin-7 show differential staining for these two cadher-ins. For example, in the nucleus of the basal optic root, thecadherin-7-positive area is a ventral subregion of thecadherin-6B-positive area. Both markers appear on bothcell bodies and on the neuropile in this area (Fig. 12). Anunexpected differential staining is also seen in the nucleuslateralis anterior of the thalamus (Fig. 11). A previouslyunreported ventral subdivision of this nucleus containsmore cadherin-7-positive structures than the dorsal one,and the same pattern is shown by cadherin-6B, althoughat a higher level of expression. In the ventral lateralgeniculate nucleus, cells in the entire nucleus expresscadherin-7 (more strongly in the plexiform layer), whereasonly the compact lamina cells express cadherin-6B. Thelatter cells show stronger expression adjacent to theinterstitial nucleus of the optic tract (ITO in Fig. 11D). Asimilar pattern is found for cadherin-7 in the superficialsynencephalic nucleus (SS in Fig. 11D).

The displaced ganglion cells of the retina and thenucleus of the basal optic root are part of the accessoryoptic system in birds (Brecha et al., 1980; Fite et al., 1981).Other components of this system also express cadherin-6Band cadherin-7, including the external pretectal nucleus(see above), the inferior olive, the paraflocculus of thecerebellum, the interstitial nucleus of Cajal (data notshown), and the oculomotor nuclei (Fig. 13). In the ventro-medial oculomotor subdivision (vm in Fig. 13D–F), largeneurons in the ventral part coexpress the two cadherins(arrowheads in Figs. 5E, 13D,E), whereas those in thedorsal part only express cadherin-6B (arrows in Figs. 5E,13D,E). The accessory (Edinger-Westphal) nucleus of theoculomotor complex, which is not connected to the nucleusof the basal optic root, does not express cadherin-6B orcadherin-7.

DISCUSSION

Previous studies on the developing chicken retina haveshown that N-, R-, and B-cadherins are differentiallyexpressed by the layers of the neural retina and by thepigment epithelium (Matsunaga et al., 1988b; Inuzuka etal., 1991; Murphy-Erdosh et al., 1994; Liu et al., 1997).N-cadherin expression was also found in the retinofugalfibers and their main projection area, the optic tectum(Redies et al., 1993; Redies and Takeichi, 1993). In thepresent work, we extend these studies and map theexpression of two additional cadherins (cadherin-6B and

Fig. 7. A: Cadherin-7 (cad 7) expression by different cell types ofthe retina at 18 days of incubation (E18). Immunofluorescence resultsare shown. The small arrowheads point at bipolar cells in the innernuclear layer (INL). The large arrowheads point at amacrine cells. Thesmall arrows point at the two neurites of a bipolar cell. The large arrowpoints at a faintly stained ganglion cell in the ganglion cell layer(GCL). B: Cadherin-7 expression in outer layers of the E18 retina. Thearrows point at dotlike immunoreactivity in the outer limiting mem-brane. Scale bars 5 20 µm in A, 10 µm in B.


cadherin-7) in the embryonic retina (E2.5–E19). We showthat all five cadherins are differentially expressed byspecific cell types and their subpopulations. Moreover,cadherin-6B and cadherin-7 are expressed by subsets ofretinal ganglion cells, their neurites, and their targetareas in the di- and mesencephalon. Some of these targetareas also express cadherin-6B and cadherin-7 and formpart of specific neural circuits in the visual system.

Cadherins are differentially expressedby retinal cell types and their subpopulations

Results from the present study show that the Mullerglial cells of the neural retina express B-cadherin. Mullercells also express N-cadherin and R-cadherin (Inuzuka etal., 1991). We could not identify any nerve cell somata orprocesses expressing B-cadherin. B-cadherin and its likelyhomologues are also expressed by the pigment epitheliumin chicken and other vertebrate species, as reported previ-ously (Murphy-Erdosh et al., 1994; Redies and Muller,1994).

The differential expression of cadherin-6B, cadherin-7,R-cadherin, and N-cadherin by neuronal cell types in theretina can be summarized as follows. The outer nuclearlayer contains photoreceptor cells that, at E14, expressN-cadherin (Figs. 2H, 6A; see also Matsunaga et al.,1988b) but not the other four cadherins. The outer plexi-

form layer is relatively uniformly stained by all fourcadherins. In the inner nuclear layer, some horizontal cellswere found to express N-cadherin (large arrow in Figs. 2H,6A). We could not clearly identify individual horizontalcells expressing any of the other cadherins. Given thedense expression pattern of cadherin-7 in this layer, wecannot exclude the possibility that cadherin-7 is expressedby horizontal cells. Cadherin-6B and cadherin-7 are ex-pressed by different subpopulations of bipolar cells thatdiffer in their average vertical position within the innernuclear layer (Figs. 2J,K,Q,R, 6C,D, 7), although there is apartial overlap. R-cadherin is also expressed by bipolar cellsomata in the inner half of the inner nuclear layer (Figs.1P, 2B,I,P, 9A) and may also be present in Muller glia inouter regions of this layer. Cadherin-6B and cadherin-7are expressed by amacrine cells, including bushy andinverted amacrine cells (Genis-Galvez et al., 1977). Bushyamacrine cells include populations expressing ChAT, tyro-sine hydroxylase, and other neurotransmitters and theirreceptors (Millar et al., 1987; Hering and Kroger, 1996).The subset of amacrine cells expressing cadherin-6B isdifferent from that expressing ChAT, but the two popula-tions occupy similar radial levels in the ganglion cell andinner nuclear layers and their neurites occupy the samesublaminae of the inner plexiform layer (Figs. 4A–C,5B,C). The more numerous cadherin-7-expressing ama-

Fig. 8. Differential expression of cadherins in the optic nerve head(NH) and optic nerve (ON) of the chicken embryo at 8.5 days ofincubation. Immunofluorescence results are shown. Sections werestained with antibodies against N-cadherin (N-cad; A), R-cadherin(R-cad; B), cadherin-6B (cad 6B; C), and cadherin-7 (cad 7; D). Results

are from consecutive sections. The arrows in D point at immunoreactiv-ity associated with optic nerve glia. The stars in A represent anartifactual space. The boxed area in B is shown at a higher magnifica-tion in Figure 9A. For other abbreviations, see list. Scale bar 5200 µm.


crine cells form a different subset, and they extend neu-rites into several additional sublayers of the inner plexi-form layer (Figs. 2K,R, 4D, 5C, 7). All of these layersexpress the synaptic vesicle-associated molecule synapsinI (Fig. 5H,I; Sudhof et al., 1989; Lu et al., 1996), which isfound in conventional synapses but not in the ribbonsynapses of the inner plexiform layer (Mandell et al.,1990). In contrast, the N- and R-cadherin immunoreactiv-ity in the inner plexiform layer is roughly complementary

to that of synapsin I (Fig. 5F,G). Therefore, the terminalsof the R-cadherin-positive bipolar cells probably are notassociated with synapses on amacrine cells expressingcadherin-6B and cadherin-7. Ganglion cells have beenshown to express N-cadherin but not R-cadherin (Matsu-naga et al., 1988b; Inuzuka et al., 1991; Redies andTakeichi, 1993). Whereas the vast majority of ganglioncells express N-cadherin during active axon outgrowth,cadherin-6B and cadherin-7 are only expressed by a subset

Fig. 9. Differential expression of cadherins by retinofugal fibersand by cell types of the retina and optic nerve head. Double-labelimmunostaining results are shown for retina at the transition to theoptic nerve head at 8.5 days of incubation. Sections were stained withantibodies against R-cadherin (R-cad; A), cadherin-6B (cad 6B; B),cadherin-7 (cad 7; C), and N-cadherin (N-cad; E,F). A nuclear stain(Hoechst 33258; Hoe) is shown in D. Pairs A–D, B–E, and C–Frepresent double-labeling immunofluorescence results from the same

section. Note the cadherin-6B-positive line of prospective displacedamacrine cells migrating through the inner plexiform layer (largearrowheads in A,B, and D). Large arrows in A and D point atR-cadherin-positive glial cell columns of the optic nerve. Small arrowsin C point at glial processes expressing cadherin-7. Small arrowheadsin B and C point at single optic nerve fibers running into the opticnerve. For other abbreviations, see list. Scale bar 5 100 µm.


of ganglion cells, including cells that are displaced into theinner nuclear layer. These cells provide retinal input to oneof the main constituents of the accessory optic system inchicken, the nucleus of the basal optic root (see below;Brecha et al., 1980; Fite et al., 1981; Britto et al., 1988). AtE14, all displaced ganglion cells identified in this studyexpressed cadherin-6B. This finding correlates with theextensive labeling of the nucleus of the basal optic root.Only about every second cell expresses cadherin-7 (Fig. 3),and immunoreactivity in the nucleus of the basal optic rootis restricted to the ventral part (Fig. 12).

In conclusion, the present study extends previous re-sults on the differential expression of N-cadherin andR-cadherin in the layers of the chicken retina (Matsunagaet al., 1988b; Inuzuka et al., 1991). We have shown thatdifferent retinal cell types and their subpopulations arecharacterized by the expression of five cadherins. It isnoteworthy that each of the cadherins examined (with theexception of B-cadherin) is expressed by subsets of neu-rons distributed throughout several layers of the neuralretina. Moreover, the plexiform layers and their sublami-nae, where the retinal neurons connect to each other, alsoshow differential cadherin expression. At least some of thecadherin immunoreactivity in the inner plexiform layers isassociated with specific types of synapses. Therefore, wespeculate that the adhesive specificity conferred by thecadherins to retinal neurons may play a role in the

formation of information processing circuits, which allow apatterned flow of information from the photoreceptors tothe ganglion cells. A function of cadherins as recognitionmolecules in neural circuit formation (Redies et al., 1993;Arndt and Redies, 1996; Suzuki et al., 1997; reviewed inRedies, 1995, 1997) and synaptogenesis (Fannon andColman, 1996; Uchida et al., 1996; reviewed in Colman,1997) has been postulated before for other brain regions.Cadherins preferentially bind to their own type, e.g.,N-cadherin to N-cadherin and cadherin-7 to cadherin-7(‘‘homophilic’’ binding). Heterophilic binding has also beendescribed (e.g., between cadherin-6B and cadherin-7), butit is generally weaker than homophilic binding (reviewedin Takeichi, 1995). It remains to be established at theultrastructural level whether the different neurons express-ing a particular cadherin in the retina are indeed function-ally and selectively connected.

Cadherins as markers for differentpopulations of optic neurites and their

functional connectivities

N-cadherin is a general marker for fibers in the opticnerve and tract. Cadherin-6B and cadherin-7 are onlyexpressed in (largely overlapping) subsets of ganglion cellsand their processes. Both cadherin-6B and cadherin-7seem to appear in early-born ganglion cells, as judged from

Fig. 10. Differential expression of cadherins in the optic chiasm(CO) of the chicken embryo at 8 days of incubation. Consecutivesections were stained with antibodies against N-cadherin (N-cad; A),R-cadherin (R-cad; B), cadherin-6B (cad 6B; C), and cadherin-7 (cad 7;D). The arrowheads in A,C, and D point at the tips of fiber bundles

coexpressing cadherin-6B, cadherin-7, and N-cadherin. The arrows inD point at optic nerve glia expressing cadherin-7. The majority ofN-cadherin-expressing optic neurites do not express cadherin-6B orcadherin-7. For other abbreviations, see list. Scale bar 5 200 µm.


their colocalization in displaced ganglion cells and fromthe course of the immunoreactive axons within the outerhalf of the retinal optic fiber layer and within the deepportion of the optic tract.

Cadherin-6B seems to be specifically related to alldisplaced (inverted) ganglion cells, which project throughthe basal optic tract into nuclei of the accessory visualsystem (Brecha et al., 1980; Fite et al., 1981; Britto et al.,

Fig. 11. Expression of cadherin-6B (cad 6B; A,D) and cadherin-7(cad 7; B,E) in retinorecipient nuclei of the diencephalon of the chickenembryo at 11 days of incubation. Results are from peroxidase immuno-histochemistry; the dark reaction product indicates cadherin expres-sion. Adjacent sections stained for Nissl substance (thio) are shown inC,F. The sections shown in A–C and D–F represent consecutive

sections. The arrowheads in A–C point at the border between thediencephalic gray matter and the optic tract (OT). The neuritesexpressing cadherin-6B and cadherin-7 represent a deep subpopula-tion of the optic tract fibers. The stars indicate artifactual spaces. Forother abbreviations, see list. Scale bar 5 200 µm.


1988). Apart from invading the neuropile of the nucleus ofthe basal optic root, these fibers also innervate the exter-nal pretectal nucleus (S. Martınez and L. Puelles, unpub-lished observations). The latter is functionally connectedto horizontal optokinetic reflexes, whereas the nucleus ofthe basal optic root is related to vertical optokineticreactions (Brecha et al., 1980; Burns and Wallman, 1981).Two other studies have shown direction-selective units inthe nucleus lentiformis, encompassing both the externalpretectal nucleus and the griseum tectale (McKenna andWallman, 1985; Winterson and Brauth, 1985).

Separate cadherin-6B-immunoreactive fibers course dor-sally to the marginal optic tract and arborize in the areapretectalis and the nucleus lateralis anterior. Area pretec-talis is related to pupillary reflex circuitry (Gamlin et al.,1984). The known connections of the nucleus lateralisanterior are largely uninformative with respect to thefunctional relevance of this nucleus (Boxer and Stanford,

1985; Ehrlich and Stuchbery, 1986; Kuenzel and Blahser,1991; Szekely et al., 1994). The accessory visual reflexesand the pupillary reflexes are originated in ganglion cellswith large receptive fields (Burns and Wallman, 1981;Morgan and Frost, 1981; Bodnarenko et al., 1988; Gamlinand Cohen, 1988).

Cadherin-7 is present in about half of the population ofthe cadherin-6B-positive displaced ganglion cells. Theterminal fields labeled with cadherin-7 include accessoryvisual nuclei. Interestingly, only roughly the ventral half ofthe nucleus of the basal optic root is cadherin-7 positive.Dorsoventral heterogeneity in this nucleus may separatecells serving up-looking from down-looking responses(Brecha et al., 1980; Burns and Wallman, 1981). Theobserved differential labeling may also be related to theretinotopic innervation because Ehrlich and Mark (1994)showed that temporal displaced ganglion cells projectdorsally and nasal ones project ventrally in this nucleus.

Fig. 12. Expression of cadherin-6B (cad 6B; A,B) and cadherin-7(cad 7; C,D) in retinorecipient nuclei of the diencephalon of thechicken embryo at 11 days of incubation. Results are from peroxidaseimmunohistochemistry; the dark reaction product indicates cadherinexpression. A section stained for Nissl substance (thio) is displayed inE and F. A,C, and E represent consecutive sections. B,D, and F

represent enlargements of the results shown in A,C, and E, respec-tively, as indicated by the boxed area in E. The optical tract (OT) ispartly missing from the sections displayed in A and C due to an artifactof preparation. For other abbreviations, see list. Scale bars 5 200 µmfor A,C,E, 100 µm for B,D,F.


Figure 13

The same argument may be used for the differentialdorsoventral staining pattern observed in the nucleuslateralis anterior: the temporal retina projects ventrallyand the nasal retina dorsally (Ehrlich and Mark, 1984). Inaddition, McKenna and Wallmann (1985) reported thatthe dorsoventral organization of directional selectivity ofunits in the nucleus of the basal optic root changes duringdevelopment, suggesting that, during a transient phase,the cells in this nucleus migrate selectively to dorsal andventral fields to reach their adult localization. Again, thismigration could be guided by the differential expression ofcadherin-6B and cadherin-7.

Curiously, although there is only weak cadherin-7 expres-sion in the ganglion cell layer itself, most retinorecipientnuclei in the di- and mesencephalon show staining for thismarker. This staining is, at least in part, due to expressionby intrinsic cells, as demonstrated by the presence ofcadherin-7 mRNA in these nuclei. Another part may bedue to invading fibers from the deep portions of the optictract, which also express cadherin-6B and cadherin-7.

As described previously, N-cadherin is also expressed bymes- and diencephalic nuclei, some of which also receivedirect retinal input (Redies et al., 1993). When comparedwith the present results for cadherin-6B and cadherin-7,the N-cadherin expression pattern is different. For ex-ample, the nuclei of the accessory optic system do notexpress N-cadherin, whereas the interstitial nucleus of theoptic tract expresses N-cadherin (C. Redies and L. Puelles,unpublished data) but not cadherin-6B and cadherin-7.R-cadherin is not expressed by any of the retinorecipient oraccessory optic nuclei (Arndt and Redies, 1996).

General conclusions

Results from the present study in the visual system arecompatible with the hypothesis that cadherins conferdifferential adhesiveness to the constituents of information-processing subcircuits. Numerous in vitro and in vivoexperiments have suggested that cadherins are likely toplay a role in developmental processes such as neuriteelongation, neurite fasciculation, axonal pathfinding, andaxonal target recognition (reviewed in Redies, 1997; Col-man, 1997). Cadherins preferentially bind to their owntype (homophilic adhesion), and cadherin-mediated differ-ential adhesiveness may therefore be involved in theformation and functional specificity of visual circuitry.Previous studies have shown that cadherins can be used asmarkers for functional connections across brain regions(Redies et al., 1993; Arndt and Redies, 1996; Suzuki et al.,1997). Results from the present study indicate that, alsoon a regional level, retinal circuitry may be specified by the

expression of cadherins, possibly even at the synaptic level(see also Fannon and Colman, 1996; Uchida et al., 1996).In many cell populations of the visual system, the expres-sion of the different cadherins is partly overlapping,especially for cadherin-6B and cadherin-7. These twocadherins bind not only to their own type but also to eachother, although more weakly (Nakagawa and Takeichi,1995). Therefore, the adhesive code specifying functionalconnections may be a combinatorial one (see also Suzuki etal., 1997), and homotypic and heterotypic interactionsbetween different types of cadherins may be involved.

ACKNOWLEDGMENTS

We are grateful to M. Ast for expert technical assistance,to Drs. E. de la Rosa, U. Drager, M. Epstein, P. Hausen,and B. Lu for antibodies, and to S. Fuhrmann and H.-D.Hofmann for discussion. This study was supported bygrants from the Land Baden-Wurttemberg NeurobiologyProgramme (to C.R.), Spanish Ministerio de RelacionesExteriores and German Academic Exchange Service (Ac-ciones Integradas grant HA1996–0149 to C.R. and L.P.),Human Frontier Science Programme (grant GR-41–95 toL.P. and M.T.), and DGICYT Governmental Grant Agency,Madrid (grant PB93–1137 to L.P.).

LITERATURE CITED

Angres, B., A.H.J. Muller, J. Kellermann, and P. Hausen (1991) Differentialexpression of two cadherins in Xenopus laevis. Development 111:829–844.

Arndt, K. and C. Redies (1996) Restricted expression of R-cadherin by brainnuclei and neural circuits of the developing chicken brain. J. Comp.Neurol. 373:373–399.

Bodnarenko, S.R., X. Rojas, and O.C. McKenna (1988) Spatial organizationof the retinal projection to the avian lentiform nucleus of the mesen-cephalon. J. Comp. Neurol. 269:431–447.

Boxer, M.I. and D. Stanford (1985) Projections to the posterior visualhyperstriatal region in the chick: An HRP study. Exp. Brain Res.57:494–498.

Brecha, N., H.J. Karten, and S.P. Hunt (1980) Projections of the nucleus ofthe basal optic root in the pigeon: An autoradiographic and horseradishperoxidase study. J. Comp. Neurol. 189:615–670.

Britto, L.R. (1989) Chemically specific retinal ganglion cells collaterize tothe pars ventralis of the lateral geniculate nucleus and optic tectum.Vis. Neurosci. 3:477–482.

Britto, L.R.G., K.T. Keyser, D.E. Hamassaki, and H.J. Karten (1988)Catecholaminergic subpopulation of retinal displaced ganglion cellsprojects to the accessory optic nucleus in the pigeon (Columba livia). J.Comp. Neurol. 269:109–117.

Burns, S. and J. Wallman (1981) Relation of single unit properties to theoculomotor function of the nucleus of the basal optic root (accessoryoptic system) in chickens. Exp. Brain Res. 42:171–180.

Colman, D.R. (1997) Neurites, synapses, and cadherins reconciled. Mol.Cell. Neurosci. 10:1–6.

Crossland, W.J., W.M. Cowan, and L.A. Rogers (1975) Studies on thedevelopment of the chick optic tectum. IV. An autoradiographic study ofthe development of retino-tectal connections. Brain Res. 91:1–23.

de la Rosa, E.J., J.F. Kayyem, J.M. Roman, Y.-D. Stierhof, W.J. Dreyer, andU. Schwarz (1990) Topographically restricted appearance in the devel-oping chick retinotectal system of Bravo, a neural surface protein:Experimental modulation by environmental cues. J. Cell Biol. 111:3087–3096.

De Long, G.R. and A.J. Coulombre (1965) Development of the retinotectaltopographic projection in the chick embryo. Exp. Neurol. 13:351–363.

Drager, U.C., D.L. Edwards, and C.J. Barnstable (1984) Antibodies againstfilamentous components in discrete cell types of the mouse retina. J.Neurosci. 4:2025–2042.

Ehrlich, D. and R. Mark (1984) An atlas of the primary visual projections inthe brain of the chick Gallus gallus. J. Comp. Neurol. 223:592–610.

Ehrlich, D. and J. Stuchbery (1986) A note on the projection from the rostralthalamus to the visual hyperstriatum of the chicken (Gallus gallus).Exp. Brain Res. 62:207–211.

Fig. 13. Cadherin expression in transverse sections through theoculomotor complex of a chicken embryo at 11 days of incubation.Sections were double stained with antibodies against cadherin-6B (cad6B; A,D) and cadherin-7 (cad-7; B,E). An adjacent section stained forNissl substance (thio) is displayed in C,F, and H. A schematic diagramindicating the location of the dorsolateral (dl), dorsomedial (dm),ventromedial (vm), and Edinger-Westphal (EW) nuclei of the oculomo-tor complex is displayed in G. The area boxed in H is shown at a highermagnification in C. The area boxed in C is shown at a highermagnification in F. Pairs A–B and D–E represent immunostainingresults from the same section. Large arrows in A–C point at theEdinger-Westphal (EW) nucleus. Small arrows in D and E point atcadherin-6B-positive neurons that do not express cadherin-7. Arrow-heads in D and E point at neurons expressing both cadherin-6B andcadherin-7. Color-coded overlays of the results shown in D and E aredisplayed in Figure 5F. For other abbreviations, see list. Scale bars 520 µm for A–C, 50 µm for D–F, 100 µm in H.


Fannon, A.M. and D.R. Colman (1996) A model for central synapticjunctional complex formation based on the differential adhesive speci-ficities of the cadherins. Neuron 17:423–434.

Fite, K.V., N. Brecha, H.J. Karten, and S. Hunt (1981) Displaced ganglioncells and the accessory optic system of pigeon. J. Comp. Neurol.195:279–288.

Fletcher, E.L. and M. Kalloniatis (1996) Neurochemical architecture of thenormal and degenerating rat retina. J. Comp. Neurol. 376:343–360.

Gamlin, P.D.R. and D.H. Cohen (1988) Retinal projections to the pretectumin the pigeon (Columba livia). J. Comp. Neurol. 269:1–17.

Gamlin, P.D.R., A. Reiner, J.T. Erichsen, H.J. Karten, and D.H. Cohen(1984) The neural substrate for the pupillary light reflex in the pigeon(Columba livia). J. Comp. Neurol. 226:523–543.

Ganzler, S.I.I. and C. Redies (1995) R-cadherin expression during nucleusformation in chicken forebrain neuromeres. J. Neurosci. 15:4157–4172.

Genis-Galvez, J.M., L. Puelles, and C. Prada (1977) Inverted (displaced)retinal amacrine cells and their embryonic development in the chick.Exp. Neurol. 56:151–157.

Goldberg, S. (1974) Studies on the mechanics of development of the visualpathways in the chick embryo. Dev. Biol. 36:24–43.

Grunwald, G.B. (1996) Cadherin cell adhesion molecules in retinal develop-ment and pathology. Prog. Retinal Eye Res. 15:363–392.

Gumbiner, B.M. (1996) Cell adhesion: The molecular basis of tissuearchitecture and morphogenesis. Cell 84:345–357.

Hamburger, V. and H. Hamilton (1951) A series of normal stages in thedevelopment of the chick embryo. J. Morphol. 88:49–92.

Hatta, K. and M. Takeichi (1986) Expression of N-cadherin adhesionmolecules associated with early morphogenetic events in chick develop-ment. Nature 320:447–449.

Hatta, K., A. Nose, A. Nagafuchi, and M. Takeichi (1988) Cloning andexpression of cDNA encoding a neural calcium-dependent cell adhesionmolecule: Its identity in the cadherin gene family. J. Cell Biol. 106:873–881.

Hering, H. and S. Kroger (1996) Formation of synaptic specializations inthe inner plexiform layer of the developing chick retina. J. Comp.Neurol. 375:393–405.

Inuzuka, H., S. Miyatani, and M. Takeichi (1991) R-cadherin: A novelCa21-dependent cell–cell adhesion molecule expressed in the retina.Neuron 7:69–79.

Karten, H.J. and W. Hodos (1967) A Stereotaxic Atlas of the Brain of thePigeon (Columba livia). Baltimore: Johns Hopkins University Press.

Kuenzel, W.J. and S. Blahser (1991) The distribution of gonadotropin-releasing hormone (GnRH) neurons and fibers throughout the chickenbrain (Gallus domesticus). Cell Tissue Res. 264:481–495.

Kuenzel, W.J. and M. Masson (1988) A Stereotaxic Atlas of the Brain of theChick (Gallus domesticus). Baltimore: The Johns Hopkins UniversityPress.

Liu, X., A. Mizoguchi, M. Takeichi, Y. Honda, and C. Ide (1997) Developmen-tal changes in the subcellular localization of R-cadherin in chick retinalpigment epithelium. Histochem. Cell Biol. 108:35–43.

Lu, B., A.J. Czernik, S. Popov, T. Wang, M.-M. Poo, and P. Greengard (1996)Expression of synapsin I correlates with maturation of the neuromuscu-lar synapse. Neuroscience 74:1087–1097.

Mandell, J.W., E. Townes-Anderson, A.J. Czernik, R. Cameron, P. Green-gard, and P. De Camilli (1990) Synapsins in the vertebrate retina:Absence from ribbon synapses and heterogeneous distribution amongconventional synapses. Neuron 5:19–33.

Martınez, S., R.M. Alvarado-Mallart, M. Martınez-de-la-Torre, and L.Puelles (1991) Retinal and tectal connections of embryonic nucleussuperficialis magnocellularis and its mature derivatives in the chick.Anat. Embryol. 183:235–243.

Matsunaga, M., K. Hatta, A. Nagafuchi, and M. Takeichi (1988a) Guidanceof optic nerve fibers by N-cadherin adhesion molecules. Nature 334:62–64.

Matsunaga, M., K. Hatta, and M. Takeichi (1988b) Role of N-cadherin celladhesion molecules in the histogenesis of neural retina. Neuron 1:289–295.

McKenna, O.C. and J. Wallman (1985) Functional postnatal changes inavian brain regions responsive to retinal slit: A 2-deoxy-D-glucosestudy. J. Neurosci. 5:330–342.

Mey, J. and S. Thanos (1992) Development of the visual system in thechick—A review. J. Hirnforsch. 33:673–702.

Millar, T.J., I. Ishimoto, I.W. Chubb, M.L. Epstein, C.D. Johnson, and I.G.Morgan (1987) Cholinergic amacrine cells of the chicken retina: A lightand electron microscope immunocytochemical study. Neurosci. 21:725–743.

Morgan, B. and B.J. Frost (1981) Visual response characteristics of neuronsin nucleus of basal optic root of pigeons. Exp. Brain Res. 42:181–188.

Murphy-Erdosh, C., E.W. Napolitano, and L.F. Reichardt (1994) Theexpression of B-cadherin during embryonic chick development. Dev.Biol. 161:107–125.

Nakagawa, S. and M. Takeichi (1995) Neural crest cell–cell adhesioncontrolled by sequential and subpopulation-specific expression of novelcadherins. Development 121:1321–1332.

Prada, C., L. Puelles, J.M. Genis-Galvez, and G. Ramirez (1987) Two modesof free migration of amacrine cell neuroblasts in the chick retina. Anat.Embryol. 175:281–287.

Prada, C., J. Puga, L. Perez-Mendez, R. Lopez, and G. Ramırez (1991)Spatial and temporal patterns of neurogenesis in the chick retina. Eur.J. Neurosci. 3:559–569.

Prada, F.A., M.M. Magalhaes, A. Coimbra, and J.M. Genis-Galvez (1989)Morphological differentiation of the Muller cell: Golgi and electronmicroscopy study in the chick retina. J. Morphol. 201:11–22.

Puelles, L., M. Guillen, and M. Martınez-de-la-Torre (1991) Observationson the fate of nucleus superficialis magnocellularis of Rendahl in theavian diencephalon, bearing on the organization and nomenclature ofneighboring retinorecipient nuclei. Anat. Embryol. 183:221–233.

Quesada, A. and J.M. Genis-Galvez (1985) Morphological and structuralstudy of landolt’s club in the chick retina. J. Morphol. 184:205–214.

Rager, G. (1980) The Development of the Retinotectal Projection in theChicken. Berlin: Springer.

Ramon y Cajal, S. (1911) Histologie du systeme nerveux de l’homme et desvertebres. Madrid: Maloine. Reprint, 1992.

Redies, C. (1995) Cadherin expression in the developing vertebrate brain:From neuromeres to brain nuclei and neural circuits. Exp. Cell Res.220:243–256.

Redies, C. (1997) Cadherins and the formation of neural circuitry in thevertebrate CNS. Cell Tissue Res. 290:405–413.

Redies, C. and H.-A.J. Muller (1994) Similarities in structure and expres-sion between mouse P-cadherin, chicken B-cadherin and frog XB/U-cadherin. Cell Adhes. Commun. 2:511–520.

Redies, C. and M. Takeichi (1993) N- and R-cadherin expression in the opticnerve of the chicken embryo. Glia 8:161–171.

Redies, C. and M. Takeichi (1996) Cadherins in the developing centralnervous system: An adhesive code for segmental and functional subdivi-sions. Dev. Biol. 180:413–423.

Redies, C., H. Inuzuka, and M. Takeichi (1992) Restricted expression of N-and R-cadherin on neurites of the developing chicken CNS. J. Neurosci.12:3525–3534.

Redies, C., K. Engelhart, and M. Takeichi (1993) Differential expression ofN- and R-cadherin in functional neuronal systems and other structuresof the developing chicken brain. J. Comp. Neurol. 333:398–416.

Redies, C., K. Arndt, and M. Ast (1997) Expression of the cell adhesionmolecule axonin-1 in neuromeres of the chicken diencephalon. J. Comp.Neurol. 381:230–252.

Rodieck, R.W. (1973) The Vertebrate Retina—Principles of Structure andFunction. San Francisco: W.H. Freeman.

Shimizu, T., K. Cox, H.J. Karten, and L.R. Britto (1994) Choleratoxinmapping of retinal projections in pigeons (Columba livia), with empha-sis on retinohypothalamic connections. Vis. Neurosci. 11:441–446.

Sudhof, T.C., A.J. Czernik, H.T. Kao, K. Takei, P.A. Johnston, A. Horiuchi,S.D. Kanazir, M.A. Wagner, M.S. Perin, P. DeCamilli, and P. Greengard(1989) Synapsins: Mosaics of shared and individual domains in a familyof synaptic vesicle phosphoproteins. Science 245:1474–1480.

Suzuki, S.C., T. Inoue, Y. Kimura, T. Tanaka, and M. Takeichi (1997)Neuronal circuits are subdivided by differential expression of type-IIclassic cadherins in postnatal mouse brains. Mol. Cell. Neurosci.9:433–448.

Szekely, A.D., M.I. Boxer, M.G. Stewart, and A. Csillag (1994) Connectivityof the lobus paraolfactorius of the domestic chicken (Gallus domesti-cus): An anterograde and retrograde pathway tracing study. J. Comp.Neurol. 348:374–393.

Takeichi, M. (1995) Morphogenetic roles of classic cadherins. Curr. Opin.Cell Biol. 7:619–627.

Uchida, N., Y. Honjo, K.R. Johnson, M.J. Wheelock, and M. Takeichi (1996)The catenin/cadherin adhesion system is localized in synaptic junctionsbordering transmitter release zones. J. Cell Biol. 135:767–779.

Winterson, B.J. and S.E. Brauth (1985) Direction-selective single units inthe nucleus lentiformis mesencephali of the pigeon (Columba livia).Exp. Brain Res. 60:215–226.


cadherin expression in the retina and retinofugal pathways of the chicken embryo

Documents