THE JOURNAL OF COMPARATIVE NEUROLOGY 373:373-399 (1996)

Restricted Expression of R-Cadherin

by Brain Nuclei and Neural Circuits
of the Developing Chicken Brain
K. ARNDT AND C. REDIES
Department of Biochemistry, Max Planck Institute for Developmental Biology,
Spemannstrasse 35/11, D-72076 Tubingen, Germany

ABSTRACT
Cadherins are a family of Ca”-dependent cell-cell adhesion molecules regulating morpho-
genesis by a preferentially homophilic binding mechanism. We have previously shown that the
expression of R-cadherin in the early chicken forebrain (embryonic days E3-E6) is restricted to
particular neuromeres or parts of neuromeres. R-cadherin-expressing neuroblasts born in
these areas accumulate in the mantle zone and aggregate in particular (pro-)nuclei (Ganzler
and Redies I1995 I J. Neurosci. 15:4157-4172). In the present study, these findings are extended
to later developmental stages (embryonic days E8, E l l , and E15). By immunohistochemical
and in situ hybridization techniques, we show that, at these stages of development, R-cadherin
expression remains restricted to particular developing gray matter regions and fiber tracts. The
R-cadherin-positive fiber tracts connect some of the R-cadherin-positive gray matter areas to
form parts of particular neural circuits in the visual, auditory, somatosensory, and motor
systems. Moreover, R-cadherin expression reflects the morphologic differentiation of gray
matter regons. As brain nuclei become morphologically more distinct, the expression of
R-cadherin shows a clearer demarcation of t h e nuclear boundaries. In addition,
R-cadherin expression in some nuclei becomes restricted to particular subregions or to clusters
of neurons. In the cerebellum, R-cadherin is expressed in parasagittal stripes. These results
suggest that R-cadherin expression reflects the functional and morphologic maturation of gray
matter structures and of information processing circuits in the embryonic chicken
brain. iWti Wiley-Liss. Inc.

Indexing terms: chick embryo, development, cell adhesion molecules, central nervous system,
cadherins

Cadherins are a family of molecules that regulate morpho-
genesis by a Ca”-dependent mechanism in a variety of
tissues of the vertebrate embryo (reviewed in Takeichi,
1988; Ranscht, 1991; Takeichi, 1991; Geiger and Ayalon,
1992; Dalseg et al., 1993; Takeichi, 1995). At least ten
cadherins are expressed in the vertebrate brain (reviewed in
Takeichi et al., 1990; Suzuki et al., 1991; Sano et al., 1993;
Redies, 1995). R-cadherin (Inuzuka et al., 1991a) is ex-
pressed in muscle, in the notochord, in the thymus, in the
pancreatic islets of Langerhans, and in the peripheral and
central nervous system (Inuzuka et al., 1991b; Redies et al.,
1992; Hutton et al., 1993; Redies et al., 1993).
The expression of R-cadherin in the early developing
chicken brain (3-6 days of incubation; E3-E6) has recently
been studied in detail (Ganzler and Redies, 1995). From the
begmning of its expression at about E2.5-E4, R-cadherin
expression is restricted to several distinct patches and
stripes at various locations throughout the brain. The
borders of some of these R-cadherin-positive areas coincide
with neuromere borders, suggesting a relation between
R-cadherin expression and the neuromeric organization of
the early embryonic brain (Ganzler and Redies, 1995).
In the R-cadherin-positive areas, R-cadherin-expressing
neurons are born. As more and more neurons accumulate
in the developing mantle layer, the R-cadherin-expressing
neurons segregate from R-cadherin-negative neurons to
form the anlagen of particular nuclei or nuclear complexes.
At the same time (E3-E6), some of the early developing
fiber tracts begin to express R-cadherin (Ganzler and
Redies, 1995).These results suggest a role for R-cadherin in
the early regional development of the chicken brain.
At a later stage ( E l l ) , R-cadherin was shown to be
expressed by parts of the tectofugal projections and their
Accepted April 23, 1996.
Address reprints to Dr. Christoph Redies, Institute for Bidom 111,
University of‘ Freiburg, Schaenzlestrasse 1, D-79104 Freiburg, Germany.
E-mail: redies(<(sun 1.biolo~~e.uni~freiburgde

( 1996 WILEY-LISS, INC.

371 K. ARNDT AND C. REDIES

target nuclei. Another cadherin, N-cadherin, is expressed
by a different component of the tectofugal system (Redies
et al., 1993). Based on these and other findings, we sug-
gested that cadherins can provide markers for parti-
cular functional systems and neural circuits in the de-
veloping brain of vertebrates (Redies et al., 1992;
Redies and Takeichi, 1993a; Redies et al., 1993; Redies,
1995).
In the present study, we used immunohistochemical
techniques more sensitive than those used previously
(Redies et al., 1993), and followed the expression of R-
cadherin in nuclei and fiber tracts at intermediate stages of
development (E8, E l l , and E15) throughout the chicken
brain. During this period, nuclei and fiber tracts become
morphologically more clearly delineated from their sur-
roundings, and complex neural circuits are established that
later process particular types of information. In view of our
previous results on the tectofugal system (Redies et al.,
1993), we asked whether, in general, the expression of
R-cadherin can be related to particular functional systems
and neural circuits in the chicken brain. Moreover, because
cadherins are morphogenetic molecules that preferentially
bind in a homophilic manner (reviewed in Takeichi, 1991,
1995),we studied the expression of R-cadherin during the
morphologic maturation of various gray and white matter
structures. We also discuss whether the types of tissue
architectures formed by R-cadherin-expressing cells are
consistent with models on the role of cell adhesion mol-
ecules in the sorting and aggregation of differentially
adherent cells (St,einberg,1963; Steinberg and Poole, 1981;

Abhreuiations

a anterior ML molecular layer of the cerebellum

AA archistriatum anterior MLd nucleus mesencephalicus lateralis, pars dorsalis
AC nucleus accumbens MnX nucleus motorius nervi vagi
A1 archistriatum intermedium N neostriatum
AL ansa lenticularis NC neostriatum caudale
ALa nucleus ansae lenticularis anterior NF neostriatum frontale
ALp nucleus ansae lenticularis posterior N1 neostriatum intermedium
Am archistriatum mediale nIX-nX nucleus nervi glossopharyngei e t nucleus motorius nervi
An nucleus angularis cochlearis vagi, pars dorsalis
Aur auricle of cerebellum nMOT nucleus marginalis tractus optici
Bas nucleus basalis nPrV nucleus principalis sensorius nervi trigemini
BCP brachium cerebellopetale nTTD nucleus tractus descendentis n e v i trigemini
no bulbus olfactorius nXII nucleus nervi hypoglossi
C caudal OM tractus occipitomesencephalicus
CA commissura anterior 0s nucleus olivaris superior
Cb cerebellum ov nucleus ovoidalis
CbL nucleus cerebellaris lateralis P posterior
CO chiasma opticum PA paleostriatum augmentatum
d dorsal PP paleostriatum primitivum
Di diencephalon PT nucleus pretectalis
DLI nucleus dorsolateralis intermedius thalami QF tractus quintofrontalis
DM nucleus dorsomedialis intercollicularis r rostra1
DMA nucleus dorsomedialis anterior thalami RL nucleus reticularis lateralis
DMK nucleus dorsomedialis hypothalami ROT nucleus rotundus
DSO decussatio supraoptica RPc nucleus reticularis parvocellularis
dT dorsal thalamus Rsd nucleus reticularis superior, pars dorsalis
E ectostriatum S nucleus tractus solitarii
EGL external granular layer of the cerebellum SC nucleus subcoeruleus
EW nucleus of Edinger-Westphal SCE stratum cellulare externum
FPL fasciculus prosencephali lateralis f lateral forebrain bundle) SGC stratum griseum centrale
fVIIIV fasciculus nervi octavii, pars vestibularis SGFS stratum griseum et fibrosum superficiale
fx fasciculus nervi v a g SL septum laterale
H habenula SLU nucleus semilunaris
Hb hindbrain SM septum mediale
HD hyperstriatum dorsale SMe stria medullaris
HM habenula medialis SNc substantia nigra, pars compacta
HP hippocampus sov shell region of nucleus ovoidalis
HT hypothalamus SPL nucleus spiriformis lateralis
HV hyperstriatum ventrale SRt nucleus subrotundus
ICO nucleus intercollicularis T nucleus triangularis
IGL internal LTanular layer of the cerebellum TD nucleus tegmenti dorsalis
IH nucleus inferioris hypothalami Tel telencephalon
Imc nucleus isthmi, pars principalis magnocellularis TeO tectum opticum
INP nucleus intrapeduncularis TO tractus olfactorius
I0 nucleus isthmo-opticus To tractus opticus
IPC nucleus isthmi, pars principalis parvocellularis TT tractus tecto-thalamicus
IPS nucleus interstitio-pretecto-subpretectalis TTI tractus tecto-isthmicus
I-x cerebellar lobules TV nucleus tegmenti ventralis
LH lamina hyperstriatica tV tectal ventricle
LHY regio lateralis hypothalami V ventral
LL lemniscus lateralis VeD nucleus vestibularis descendens
LLd nucleus lemnisci lateralis, pars dorsalis v I11 third ventricle
LI,I nucleus lemnisci lateralis, pars intermedia v IV fourth ventricle
LMD lamina medullaris dorsalis VeL nucleus vestibularis lateralis
LOC locus coeruleus VeM nucleus vestibularis medialis
LPO lobus parolfactorius VMN nucleus ventromedialis hypothalami
1v lateral ventricle vT ventral thalamus
Mes mesencephalon
K-CADHERIN EXPRESSION IN CHICKEN BRAIN 375

Glazier and Graner, 1993; Graner, 1993; Graner and was for 1 hour followed by incubation with the secondary
Sawada, 1993). antibodies for 30 minutes at room temperature. For double-
label immunostaining with antibodies against N- or R-
cadherin, respectively, and neurofilament, previously pub-
MATERIALS AND METHODS lished procedures were followed (Redies et al., 1992; Redies
Animals et al., 1993). After treatment with the antibodies, sections
Fertilized eggs from Hisex chicken (Gallus domesticus) were counterstained with the nuclear dye Hoechst 33258
were purchased from local farms and incubated at 37°C and (Sigma, St. Louis, MO) to visualize cellular nuclei. Finally,
65% humidity in a forced-draft incubator. Staging of the the sections were mounted in 90% glycerolil0T TBS
embryos was according to Hamburger and Hamilton (1951). supplemented by phenylendiamine to prevent photobleach-
The following stages were used: E8 (stage 341, E9.5 (stage ing. Fluorescence was visualized under an epifluorescence
35), E l l (stage 37), and E l 5 (stage 41). microscope (Axioplan, Zeiss, Oberkochem, Germany). For
neuroanatomic orientation, sections neighboring the immu-
Antibodies nostained sections were Nissl-stained with thionin, as
So we could visualize R-cadherin protein, the monoclonal described previously (Redies et al., 1993). All thionin stains
mouse antibody RCD-2 raised against chicken R-cadherin shown in the figures are brightfield photomicrographs.
fusion protein (Redies et al., 1992) was used. Previous Neuroanatomic structures were identified on the basis of
results from our laboratory show that this antibody specifi- an atlas of the adult chicken brain (Kuenzel and Masson,
19881, and with few exceptions, the nomenclature of that
cally reacts with R-cadherin. Immunostaining of mouse
publication was used. In addition, various neuroanatomic
neuroblastoma cells transfected with chicken R-cadherin
publications on specific regions of the chicken brain were
cDNA results in the cell-cell border staining, which is
characteristic for most cadherins, whereas untransfected consulted.
neuroblastoma cells or cells expressing other mouse and In situ hybridization
chicken cadherins are not stained with this antibody (Redies
and Takeichi, 199313). In Western blotting, the antibody Procedures were described in detail previously (Redies et
recognizes a protein of a size similar to that of R-cadherin al., 1993; Giinzler and Redies, 1995). The pBluescript (Strata-
(about 120 kDa; Inuzuka et al., 1991a). By immunostaining gene, La Jolla, Ca) DNA vector pRcad was used as a template to
of N-cadherin-expressingcells (Redies and Takeichi, 199313) produce RNA probes. This vector (a kind gift of M. Takeichi)
or by Western blotting of extracts from these cells, the contains the full-length cDNA for chicken R-cadherin (Inu-
antibody does not recognize N-cadherin, the chicken cad- zuka et al., 1991a). Sense and antisense digoxigenin-labeled
herin with the highest degree of sequence homology to RNAs were synthesized by using commercially available kits
R-cadherin (Inuzuka et al., 1991a). (Stratagene and Boehringer Mannheim, Mannheim, Ger-
So we could visualize fiber tracts, neurites were stained many) and alkaline hydrolyzed into 100-300-bpfragments.
with monoclonal mouse antibody 29B8 against the 160-kDa Three complete series of 25-pm-thick consecutive frontal
subunit of chicken neurofilament (Hatta et al., 1987; kind and transverse cryostat sections through the brain of E 11
gift of H. Fujisawa). For detection of first antibodies, and E l 5 chicken embryos were obtained as described before
appropriate secondary antibodies labeled with fluorescein, (Redies et al., 1993). Sections were spaced about 150 pm
cyanine-3, and biotin were commercially obtained (Jackson apart. They were permeabilized by treatment with protein-
ImmunoResearch, West Grove, PA). So we could visualize ase K. Nonspecific RNA binding sites were saturated with
biotinylated secondary antibody, fluorescein-labeled strep- acetic anhydride. The sections were hybridized with sense
tavidine was used (Jackson ImmunoResearch). and antisense digoxigenin-labeled RNA probes overnight a t
55°C. The sections were then washed in 5 x standard saline
Immunostaining procedures citrate (SSC) at 55°C for 10 minutes to remove coverslips
Procedures are described in detail elsewhere (Redies et and in 50% formamidei2 x SSC at 60°C for 1 hour. Single-
al., 1992; Redies et al., 1993). Briefly, heads from E8-EI5 stranded RNA was digested by RNAse A treatment. Subse-
chicken embryos were fixed for 1-3 hours at 4°C by submer- quently, the sections were washed in 50%,formamide/Z x
sion in 4% formaldehyde dissolved in Hanks’ balanced SSC at 60°C for 40 minutes, in 2x SSC at 55°C for 30
saline solution (HBSS) supplemented with 1 mM Ca*+ and minutes, and in 0 . 1 ~SSC at RT for 30 minutes. Digoxi-
1 mM Mg2+. Subsequently, heads were incubated in a genin was detected with anti-digoxigenin alkaline phospha-
graded series of sucrose solutions (12.18% sucrose), embed- tase-conjugated Fab fragments followed by a reaction using
ded in Tissue Tek 0.C.T medium (Miles, Elkhart, IN), and X-phosphate and nitroblue tetrazolium salt as substrates.
frozen in liquid Na. Sections were dehydrated, embedded in Entellan (Merck,
So we could obtain a complete overview of R-cadherin Darmstadt, Germany), and viewed under a transmission light
expression in the E8, E l l , and E l 5 chicken brain, at least microscope (Zeiss Axioplan). All in situ hybridization results
two series each of frontal and transverse sections were shown in the figures are brightfield photomicrographs.
obtained in a refrigerated microtome for each developmen-
tal stage. Sections of 10-20-pm thickness were mounted on RESULTS
coated glass slides and air dried. Sections were postfixed for
10 minutes at 4°C in formaldehyde/HBSS, washed in General observations
Tris-buffered saline (pH 7.6) containing 1mM CaClg (TBS) At the developmental stages analyzed in this study ( 8
and incubated for 20 minutes at room temperature in a days, 11 days, and 15 days of incubation; E8, E l l , E15),
solution containing 5% skimmed milk, 0.3% Triton X-100, R-cadherin is expressed in a restricted fashion by particular
and 0.04% sodium azide in TBS (“blocking solution”) to gray and white matter structures throughout the embry-
block nonspecific binding of antibodies. Incubation with the onic chicken brain. Generally, the structures expressing
first antibodies appropriately diluted in blocking solution R-cadherin remained the same, but developmental changes
 376
in the expression pattern were observed within the struc-
tures. At the earliest stage (ES), the morphology of most
gray and white matter regions is poorly defined, and
R-cadherin is diffusely expressed in several brain regions.
As brain architecture matures and individual neuroana-
tomic structures become more distinct between E8 and
E15, the R-cadherin-positive areas more clearly stand out
from their R-cadherin-negative surroundings. In general,
three types of R-cadherin expression by individual brain
nuclei can be distinguished: (1)expression by most, if not
all, cells of an entire nucleus; (2) expression by a cluster of
cells within a nucleus; and (3) expression by a few R-
cadherin-positive cells scattered throughout a nucleus. The
staining of fiber tracts with the R-cadherin antibody was
generally weaker and less distinct than that of gray matter.
R-cadherin expression was most clearly observed in parts of
some large tracts composed of prominent fiber bundles.
Whether some of the more diffusely distributed fiber projec-
tions within the brain also express this molecule was
difficult to assess.
Besides gray and white matter, other brain tissues also
express R-cadherin (e.g., radial glia and ependymal deriva-
tives such as the floor plate, roof plate, and the choroid
plexus) (Inuzuka et al., 1991b; Lagunowich et al., 1992;
Redies et al., 1993). These structures were not examined in
the present study.
Comprehensive overview
of R-cadherin expression
The gray and white matter areas expressing R-cadherin
at E8, E l l , and E l 5 in the embryonic chicken brain were
systematically mapped by immunohistochemistry in at
least five series of frontal and transverse sections for each
developmental stage. The immunostaining results were
compared with results from in situ hybridization of similar
series of sections (two series for E l l and one series for
E15). With few exceptions (see below), gray matter areas
expressing R-cadherin mRNA were also found to be R-
cadherin-positive by immunostaining.
Table 1 gives a listing of all R-cadherin-positive struc-
tures identified at each stage, together with the type of
immunostaining observed. R-cadherin-positive structures
are found in all major subdivisions of the brain. At the
earliest stage examined (ES), nuclei in many regions are
still present in a common anlage and could therefore not be
identified on an individual basis. At E 11, R-cadherin expres-
sion is most prominent and neuroanatomic structures were
identified by their location and morphology, which is simi-
lar to those of the adult brain. At E15, basically the same
structures express R-cadherin as at E l 1, although the
staining pattern changes, as described above. The expres-
sion of R-cadherin in most of the structures listed in Table 1
persists at least to the hatching stage, but this expression
was not studied in detail.
For one brain at each stage, immunostaining results from
a rostral-to-caudal series of frontal sections were schemati-
cally represented in diagrams. For this purpose, sections
immunostained for R-cadherin and adjacent Nissl stains
were viewed and compared under the microscope. R-
cadherin-positive structures were identified and marked on
photographs of the adjacent Nissl stains. Based on these
photographs, schematic representations of the R-cadherin-
positive structures were produced by scanning the outlines
of the brain sections and the R-cadherin-positive regions by
a computer. Symbols were introduced into these computer-
K. ARNDT AND C. REDIES
generated diagrams to represent the different types of
staining observed. An example for this type of analysis is
given in Figure 1, which shows an immunostained frontal
section from an E l l brain (Fig. lB), together with the
corresponding Nissl stain (Fig. 1A) and the schematic
diagram of the immunostaining results (Fig. 1C). In the
following, other schematic diagrams from the same series of
E l 1 brain sections are shown. These diagrams are used to
show R-cadherin-positive brain nuclei and fiber tracts of
each of the major subdivisions of the E l l brain. Their level
and orientation are represented in Figure 2. We also
describe examples of developmental changes of R-cadherin
expression from E8 to E l 5 observed in various neuroana-
tomic structures.
Telencephalon. Figure 3 shows R-cadherin expression
in the telencephalon, which consists of relatively large
masses of cells. The expression of R-cadherin is restricted to
particular regions. The bulbus olfactorius (BO) and several
components of the pallium and subpallium of the telencepha-
Ion express R-cadherin. Of the pallial elements, parts of the
neostriatum (N, NC, NF) (Fig. 3A-F), the ectostriatum (E)
(Fig. 3B,D), the hyperstriatum ventrale (HV) (Fig. 3A-F),
the archistriatum (AA,AI, Am) (Fig. 3C-F), and the cortex
piriformis are R-cadherin positive. Large areas of the
subpallial elements, the medial and caudal areas of the
paleostriatum augmentatum (PA) (Fig. 3C,F), the paleo-
striatum primitivum (PP)(Fig. 3D,E), the nucleus intrape-
duncularis (INP) (Fig. 3D), and the nucleus accumbens (Ac)
(Fig. 3C,D) express R-cadherin. The nucleus basalis (Bas)
(Fig. 3A,B), the septa1 nuclei (SM and SL)(Fig. 3B,D,E),
and the nucleus taeniae partially express R-cadherin. The
lobus parolfactorius (LPO) (Fig. 3A,B) contains only a few
small R-cadherin-positive regons.
Of the fiber tracts in the telencephalon, the tractus
olfactorius (TO) (Fig. 3A-C), the stria medullaris (SMe)
(Fig. 3D-F), parts of the anterior commissure (CA) (Fig.
3D), the tractus occipitomesencephalicus (OM) (Fig. 3F),
and scattered fascicles between the various R-cadherin-
positive pallial and subpallial elements express this mol-
ecule.
Some of the R-cadherin-positive cell masses show dis-
tinct morphologic boundaries that coincide with boundaries
of R-cadherin expression. For example, R-cadherin expres-
sion in the ectostriatum (E) falls off at the lamina medul-
laris dorsalis (LMD) (Fig. 3D). The transition between
other cell masses is more gradual, and R-cadherin expres-
sion changes gradually in these areas (e.g., within the
neostriatum IN]) (Fig. 3A-F).
Figure 4 shows a comparison of morphologic (Fig. 4A), in
situ hybridization (Fig. 4B), and immunostaining data (Fig.
4C) for adjacent frontal sections from an E l 5 brain. The
level of these sections approximately correspond to the level
of the diagrams shown in Figure 3D,E. In general, there is
good agreement between the location of R-cadherin tran-
script and immunostaining signals, except for the hippocam-
pus (Hp), which shows R-cadherin transcript but weak
immunostaining signal.
Diencephalon. R-cadherin-positive structures are found
in the epithalamus, in the dorsal and ventral thalamus, and
in the hypothalamus (Figs. 1, 5). Nuclei that express R-
cadherin in their entirety are the habenula medialis (HM)
of the epithalamus (Figs. 1, 5A), the nucleus subrotundus
(SRt) (Fig. 5B), the nucleus suprarotundus, the nucleus
triangularis (TI (Fig. 5B-D), the shell region surrounding
the nucleus ovoidalis (sOV)(Figs. 1,5B),the nuclei dorsolat-
 R-CADHERIN EXPRESSION IN CHICKEN BRAIN 377
'I'ABLE 1 Comprehensive Overview of R-cadherin Expression a t Three TABLE 1. Comprehensive Overview of R-cadherin Expression a t Three
Developmental Stages' Developmental Stages' (continued 1
Developmental Developmental
stages stapes

Brain r e g i o n E8 Ell El5 Brain region E8 El1 El5

Trlt~ncephdilun Nucleus motonus nerw vagi 1 -

Rulhus olfactorius Nucleus nervi hypoglossi * I * % "*

Palhum Nucleus tractus solitarii I * I * * f

Nucleus rrticularis parvocellularis

Neostriatum frontale
K'eostriatum Intermedium r Nucleus reticularis lateralis
1 *.l
I d . ,
I

Neostriatum caudale Nucleus raphe <. >

Hyperstriat u m ventral? Cranial nerve Fascicles
Ectristriatum *x.1
Fasciculus nervi octavii, pars vestibularis
Archistriaturn anterior Fasciculus nervi glossopharyngei et nervi va~e
Archistriaturn intermedium Fiber connections
Archistriaturn mediale Tractus olfactorius <
Cortex piritbrmis
'Suherculum olFactorium
Suhpallium
Nuclpus basalis
t **
Ansa lenticularis
Stria medullaris
Fasciculus prosencephali lateralis
Tractus quintdrontalis \'
Lobus parr~lfactorius Tractus occipitomesenceph;tllcus
Paleustriatum primitivum Tractus tectii-isthmicus
Paleortriatum augmentatum Tractus tecto-thalamicus
Nucleus intrapeduncularis **'$ 1 1
Brachium crrpbellopetale
Nucleus accumbens Corpus trapezoideum
Septum mediale Lemniscus lateralis i.
Septum laterale
Nucleus taeniw L
Diencephdcin
Epithalamus
I**
Hahenula mpdialis 1*
, most, i f not all, cells in a nucleus or region express R-cadhenn. s. R-cadherin
Dorsal thalamus expression by parts of a nucleus or fiber tract. ,-, could not be identified.
Nucleus dorsomedialis anterior thalami
Nucleus dorsolateralis posterior thalami
Kiuclrus dorsolateralis intermedius thdarni
r 'R-cddherin expression hy particular layers.
'Not demarcated.
'ion by scattered cells (e.g., see F I ~8S, 121.
Nucleus reticularis superior, pars dorsalis 1I -ion hy clusters ofcells 1e.g..see Fig. 7C,Dl.
Nucleus rotundus *i hR-cadhcrin expression in lonwtudinal stripes (see Figs. 16-18,.
Nucleus triangularis :R-cadherin expression only by neurltes
Sucleus suprarotundus **I

Nucleus subrotundus ** **
Nucleus m a r p n a l i s tractus uptici
Nucleus woidalis *I erales intermedius (DLI) et posterior, and the nucleus
Shell region ril'the nucleus ovoidalis
*I
spiriformis lateralis (SpL) (Fig. 5D) of the thalamus. Sev-
N u c l e u s prctectali
L
eral thalamic nuclei can be subdivided into R-cadherin-
Ventral thalamus
**'I
positive and R-cadherin-negativecompartments. These nu-
S t r a t u m cellulare externum
N u c l r u s ansae lenticularis anterior ** "4 clei are the nucleus dorsomedialis anterior (DMA) (Figs. I,
Nucleus ansae lmticularis posterior ** *.I 5A), the nucleus reticularis superior, pars dorsalis (Rsd)
Nuclrus intPrstitio-pretect~,-subpretectalis **1

Hypothalamus
(Fig. l), the nucleus ovoidalis (Ov) (at E15), the nucleus
ansae lenticularis anterior (ALa) (Figs. 1,5B,C),the nucleus
r **
interstitio-pretecto-subpretectalis (IPS),the nucleus rotun-

Mesencephalon
L **
**
** dus (ROT) (Figs. 1, 5B-D), and the nucleus marginalis of
the optic tract (nMOT), which surrounds the nucleus
Nucleus isthmi. pars principalis magnocellularis *3 i* **
Sucleus Isthrni. pars principalis parvocellularis ** I* rotundus like a shell (Figs. 1,5B,C).
Nucleus isthrno-opticus **:, *- **2 In the hypothalamus, some of the caudal regions express
Nucleus semilunaris I* XI **
Nucleus mesencephalicus lateralis, pars dorsalis *i R-cadherin relatively uniformly, but these regions are not
Nuc1t.u~intercallicularis **'/ *, as well demarcated as those in the thalamus. The R-
Nucleus dorsomedialis mtercollicularis *I /I 1*
cadherin-positive regions of the hypothalamus are the
Suhstantia nigra, pars compacta **./

Nucleus interpfduncularis 0 ** I* nucleus dorsomedialis (DMN) (Kuenzel and van Tien-

Nucleus o S Edinger-Westphal ** hoven, 1982) (Fig. 5D), the nucleus ventromedialis (VMN)
Nuclrus oculomotorius 6% , n
(Fig. 5B-D), the nucleus inferioris hypothalami (IH), and
'rectum o p t x u m * I x1 *"
Cerebellum parts of the regio lateralis hypothalami (LHy) (Figs. 1,
Nucleus cerehellaris lateralis 0 5C,D).
Lobules I-X 0 Lh

Rhomhencephalon Of the diencephalic fiber tracts, R-cadherin is expressed

Locus coeruleus
Nucleus teamenti ventralis
Nucleus principalis sensorius nervi trigemini
Nucleus a n p l a r i s cochlearis
Nucleuh olivaris superior
Nuclrus magnocellularis cochlearts
Nucltws laminaris
Nuclrus ncrvi glrissopharyngei et nucleus moto-
rius newt v;i&q. pars dorsalis
**'I
by the stria medullaris (SMe) (Figs. 1,3D-F, 5A) and by the
ansa lenticularis (AL) (Figs. 1,5A-D).Moreover, R-cadherin-
positive fiber fascicIes in the diencephalon constitute minor
0
parts of the tractus tecto-thalamicus (TT) (Fig. 5D), of the
0 decussatio supraoptica (DSO)(Fig. 5B,C), and of the lateral
** 3
** forebrain bundle (FPL) (Fig. 5A).
In the following, we discuss some of these structures in
I*!
more detail. Figures 6 and 7 show the nucleus rotundus
I* (ROT), a n example of a nucleus in which R-cadherin
**/
expression becomes restricted to particular clusters of cells.
')
In Figure 6, R-cadherin immunostaining results are shown
on the right-hand side, and for neuroanatomic orientation,
I*
Nissl stains of neighboring sections are shown on the
 d
I
v
Fig. 1. Representative example of the primary immunostaining sections. Immunostaining results were graphically transferred onto t h e
data used to generate t h e schematic displays of R-cadherin expression adjacent Nissl stain for identification of t h e R-cadherin-positive neurn-
in Figures 3 , 5,11, and 15. Frontal sections of the rostra1 diencephalic anatomic structures. C: A schematical representation of t h e results
area from a n E l l embryo stained for Nissl substance (A)and immuno- from thissection. Ahhreviationsare listed separately. Scale bar = 500 pm.
stained for R-cadherin ( B ) are shown. A and B represent adjacent
R-CADHERIN EXPRESSION IN CHICKEN BRAIN
Figure 1 (Continued. 1
U I - U I
A E9.5, the anlage of the nucleus rotundus and adjacent
Fig. 2. Lateral view of the E l l chicken brain, summarizing levels
and orientations of the frontal sections displayed in Figures 1, 3, 5, 11,
and 15. The respective figure numbers and panel letters are indicated
on top. Abbreviations are listed separately.
left-hand side. At E8, the anlage of this nucleus cannot be
clearly demarcated from the rest of the dorsal thalamus
(dT) (Fig. 6A), and R-cadherin is expressed relatively
379
diffusely by the entire dorsal thalamic area (Fig. 6B). At
nuclei are more clearly discernible (Fig. 6C), and the
borders of R-cadherin expression in the nucleus rotundus
coincide with the borders of this nucleus (Fig. 6D). Within
the nucleus rotundus, R-cadherin expression is restricted to
clusters of cells located dorsomedially and dorsolaterally.
These clusters are even more prominent a t E l l (Fig. 6E,F).
At E15, the R-cadherin-expressing cells can be clearly
distinguished by their relatively large cellular nuclei and
their highly branched processes, which are restricted to
specific nuclear subregions (Fig. 7 ) .
A comparison of in situ hybridization (Fig. 7B) and
immunstaining results (Fig. 7C) again shows a close agree-
ment of the two sets of data. However, in the nucleus
rotundus, a type of cell was found that expresses relatively
high levels of R-cadherin transcript but low levels of
protein. The cells of this type are widely scattered through-
out the nucleus and do not show R-cadherin-positive
neurites.
The nucleus pretectalis is an example of a nucleus in
which a minority of cells expresses R-cadherin a t E l 5 (Fig.
8). The vast majority of cells in this densely packed nucleus
expresses another cadherin, N-cadherin (Redies et al.,
1993). At a higher magnification (Fig. 8D), loosely arranged
R-cadherin-positive nerve cell bodies and their processes
can be seen. These processes extend like a web over the
entire nucleus but are clearly confined to it (Fig. 8C). The in
 B

d
I
V

Figure 3
R-CADHERIN EXPRESSION IN CHICKEN BRAIN 381

Fig. 4. Frontal sections of the forebrain at the level of the paleostria-

tal complex from an E l 5 embryo. The panels represent consecutive
sections stained for Nissl substance (A),R-cadherin transcript (B),and
R-cadherin protein (C).Abbreviations are listed separately. Scale bar =
500 pm.

situ hybridization results (Fig. 8B) show that the cell Figure 9 shows an example of an R-cadherin-positive
bodies are concentrated in the inner part of the nucleus fiber tract, the stria medullaris (SMe) of the epithalamus.
(Fig. 8B). In this figure, R-cadherin immunostaining results are
shown on the right side and neurites revealed by anti-
neurofilament immunohistochemistry on the left side for
Fig. 3 . Schematic representation of R-cadherin expression in fron- neuroanatomic orientation. At ~ 8 the , stria medullaris
tal sections of the forebrain displayed in a rostral-to-caudal sequence.
Sections are at levels of the nucleus basalis (A,B), of the rostra1 expresses less R-cadherin than the habenular area (H) by
paleostriatal complex (C,D),and of caudal paleostriatal complex (E,F), which it is surrounded (Fig. 9A,B). At E15, the stria
as shown in Figure 2. In each section, the midline is on the right-hand medullaris exDresses R-cadherin in its oval-shaDed Dart
1 1

side. An example of the primary data is shown in Figure 1. R-cadherin composed of irominent fiber bundles (arrowhead in Fig.
expression by gray matter regions is indicated by dotted areas. More
9 ~ )Of, the habenular nuclei, only the habenula medialis
densely dotted areas exhibit stronaer immunostainina. Black sauares __.. . - .. - ~~ ~

represent R-cadherin-positive axon fascicles. Abbrevitions are-listed (HM) remains fl-cadherin Positive (Fig. 9D). XostrallY,
separately. Scale bars = 500 pm. some of the R-cadherin-positive fascicles of the stria medul-
382
Fig. 5. Schematic representation of R-cadherin expression in fron-
tal sections of an E l l diencephalon displayed in a rostral-to-caudal
sequence. Sections are at levels of the habenula medialis (A) and of the
nucleus rotundus iB-D), a s shown in Figure 2 . In each section, the
midline is on the right-hand side. An example of the primary data is
laris can be followed to the R-cadherin-positive part of the
area septalis and, caudally, to the habenula medialis.
Figure 10 shows the ansa lenticularis (AL). At E8, the
ansa lenticularis is located ventromedially in the lateral
forebrain bundle (FPL) (Fig. 10A,B) (Kuhlenbeck, 1937).
Only the ventromedial part of the fiber tract shows R-
cadherin expression. At E15, the ansa lenticularis forms a
separate fiber tract. In the ventromedial part of this tract,
neurites strongly express R-cadherin, and dorsally, the
K. ARNDT AND C. REDIES
shown in Figure 1. R-cadherin expression by gray matter regions is
indicated by dotted areas. More densely dotted areas exhibit stronger
immunostaining. Black squares represent R-cadherin-positive axon
fascicles. Abbreviations are listed separately. Scale bar = 500 pm.
expression of this molecule gradually diminishes. Rostrally,
some of the R-cadherin-positive fascicles of the ansa lenticu-
laris can be followed to the R-cadherin-positive paleostria-
tum primitivum (PP) and, caudally, to the nuclei ansae
lenticulares anterior (ALa) (Fig. 10) et posterior (ALP) and
to the nucleus spiriformis lateralis (SpL).
Mesencephalon. R-cadherin-positive structures of the
mesencephalon are shown in Figure 11. They include nuclei
in the isthmic area (the nucleus isthmi, partes magnocellu-
R-CADHERIN EXPRESSION IN CHICKEN BRAIN 383

Fig. 6. Frontal sections of the dorsal thalamus from E8 (A,B), E9.5 (C,D), and E l l (E,F) embryos stained for Nissl
substance (A,C,E) and immunostained for R-cadherin (B,D,F).The panel pairs A,B, C,D, and E,F represent adjacent sections.
In each section, the midline is on the left side. Abbreviations are listed separately. Scale bars = 200 &m.

laris et parvocellularis, the nucleus semilunaris, and the
nucleus isthmo-opticus) (Ipc, Imc, SLu, and 10) (Fig.
11D,E) and the posterior nucleus of the ansa lenticularis
(ALP) (Fig. 11A), which resembles the anterior nucleus in
its immunostaining pattern. R-cadherin-positive regions
are also found in the nucleus mesencephalicus lateralis,
pars dorsalis (MLd), and of the nucleus intercollicularis
(ICo) (Fig. 1lC-E), especially in its nucleus dorsomedialis
(DM) (Fig. 11C,D).Diffusely scattered R-cadherin-positive
cells are seen in the pars compacta of the substantia nigra
(SNc) (Fig. 11D). Moreover, there is weak expression by
neurons in the Edinger-Westphal nucleus (EW) (Fig. 11D)
and the nucleus oculomotorius (nIII). Here, the surface
membrane staining of the relatively large perikarya is
384 K. ARNDT AND C. REDIES

Fig. 7. Frontal sections of the nucleus rotundus from an E l 5 embryo stained for Nissl substance (A), R-cadherin
transcript (Bl, and R-cadherin protein (Cl. The three panels represent consecutive sections. D represents an enlargement of
the region, indicated by the hatched box in C. The midline is on the left side. The asterisk in B indicates a blood vessel.
Abbreviations are listed separately. Scale bars = 300 krn in A-C, 20 pm in D.

especially evident (Fig. 12). The different layers of the
tectum also express R-cadherin, as described previously
(Redies et al., 1993).
R-cadherin-positive fiber tracts in the mesencephalon
are the ansa lenticularis (AL) (Fig. llA-D), the tractus
tecto-isthmicus (TTI) (Fig. 1l E ) , the tractus quinto-
frontalis (QF),and weakly stained fascicles likely to repre-
sent parts of the medial forebrain bundle and the tractus
occipitomesencephalicus (OM1.
Figure 13 shows the nucleus isthmi, pars parvocellularis
(Ipc),an example of a nucleus in which most, if not all, cells
express R-cadherin. At E8, the anlage of this nucleus
represents a relatively poorly demarcated mass of cells in
the isthmic area (Fig. 13A). R-cadherin expression is promi-
nent in the anlage but not restricted to it (Fig. 13B).
R-cadherin-positive nerve fascicles of the tractus tecto-
isthmicus (TTI), which emerge from this area, are also
seen. At E l 1, the nucleus isthmi, pars parvocellularis,
consists of densely packed cells and is clearly demarcated
from its surroundings (Fig. 13C). R-cadherin expression
falls off a t the borders of the nucleus (Fig. 13D), except at
locations where the nucleus is adjacent to the R-cadherin-
positive tractus tecto-isthmicus,At E 15, R-cadherin expres-
sion by the neurites of the tractus tecto-isthmicus has
decreased to low levels (Fig. 13E,F). The nucleus isthmi,
pars magnocellularis (Imc),also expresses R-cadherin (Fig.
13C-F), but its staining is less dense than that of the
nucleus isthmi, pars parvocellularis.
Figure 14 shows the nucleus isthmo-opticus (IO), an
example of a laminated nucleus with heterogeneous R-
cadherin expression. The cells in this nucleus are evenly
distributed at E 11, and R-cadherin expression is relatively
uniform (Fig. 14A,B).At E15, when the neuropil and nerve
cell bodies have segregated from one another into different
layers (Fig. 14C,D), the neuropil shows stronger R-cadherin
expression than the layers containing the cell bodies (Fig.
14E).
Cerebellum and associated nuclei. R-cadherin expres-
sion is observed in the lobules of the cerebellum (Cb),in the
nucleus cerebellaris lateralis (CbL),and on fascicles of the
brachium cerebellopetale (BCP) (Fig. 15A,B).
In the cerebellar anlage, only weak expression is observed
in the external granular layer and the ependymal layer at
E8. Figure 16 shows a comparison of in situ hybridization
and immunostaining results for the cerebellarihindbrain
region of an E l l embryo. No obvious mismatches were
found. Staining intensity is strong in the cerebellum, where
R-cadherin is expressed in a stripe-like pattern in the
lobules. In the auricle and unfoliated parts of the cerebellar
cortex, staining is more diffuse.
Figure 17 shows that the stripe-like expression of R-
cadherin in the lobules is most prominent in the cell-sparse
molecular layer (ML) and extends into the developing
internal granular layer (IGL). Figure 18B shows an ante-
rior view of the E l l cerebellum, as indicated by the arrows
in Figure 18A. The cerebellar stripes extend in a parasagit-
 K-CADHERIN EXPRESSION IN CHICKEN BRAIN 385

Fig. 8. Frontal sections of the nucleus pretectalis from an E l 5 embryo stained for Nissl substance (A),
R-cadherin transcript (B),and R-cadherin protein (C).The three panels represent consecutive sections. D
represents an enlargement of the region, indicated by the hatched box in C. The midline is on the left side.
Abbreviations are listed separately. Scale bars = 100 pm in A-C, 20 pm in D.

tal plane over several lobules (see also Fig. 17A,B). Each
lobule contains two to three stripes on both sides of the
midline, with a small stripe located close to the midline and
one or two broader stripes located in a more lateral position
(Figs. 17A,B, 18B). The R-cadherin-positive stripes extend
through all lobules, but they are particularly strong in
lobules IV-VII. At E15, the intensity of the stripe-like
expression of the cadherin in the lobules has diminished. A
more diffuse staining persists in the internal granular layer
(data not shown).
Rhombencephalon. In the anterior rhombencephalon,
R-cadherin is expressed by the dorsal part of the nucleus
principalis sensorius nervi trigemini (nPrV), by parts of the
nucleus lemnisci lateralis (LLd and LLi) (Fig. 161, by
scattered cells in the locus coeruleus (LoC) and subcoer-
uleus, partes dorsalis et ventralis (SC) (Fig. 16), by the
nuclei tegmenti dorsalis ventralis (TD, TV) (Fig. 16) and by
an intermediate part of the nucleus raphe. In the vestibular
complex, R-cadherin-positive areas include several cell
clusters in the nucleus vestibularis lateralis, dorsal parts of
the nucleus vestibularis medialis (VeM) (Fig. 15B), and
mediocaudal regions of the nucleus vestibularis descendens
(VeD) (Fig. 15C). R-cadherin expression is also found in
lateral parts of the nucleus angularis cochlearis (An) (Fig.
E B ) , the nucleus magnocellularis cochlearis, and the
nucleus laminaris. Here, the neuropil is R-cadherin positive
at E15. Interestingly, the cells of the nucleus laminaris
themselves do not express R-cadherin mRNA (data not
shown).
Moreover, the entire nucleus olivaris superior (0s)(Fig.
15A,B), small parts of the nucleus tractus descendentis
nervi trigemini (nTTD) (Fig. 15A), and parts of the nucleus
reticularis lateralis (RL) (Fig. 15D) are R-cadherin positive.
Small cells expressing R-cadherin are scattered throughout
 K. ARNDT AND C. REDIES

Fig. 9. Frontal sections of the epithalamus from E8 (A,B) and E l 5 (C,D)embryos immunostained for
neurofilament (A,C) and for R-cadherin (B,D) showing the stria medullaris. The panel pairs A,B and C,D
represent double-staining results from the same sections, respectively. In each section, the midline is on the
left side. Abbreviations are listed separately. Scale bars = 100 IJ-m.

the ventral part of the rhombencephalon at the level of the
nucleus olivaris superior (0s);they probably belong to the
nucleus reticularis parvocellularis (Rpc) (Fig. 15A). Of the
cranial nerve nuclei, the nucleus nervi glossopharyngei et
nucleus motorius nervi vagi, pars dorsalis (nIX and nX)
(Fig. 15C), the nucleus motorius nervi vagi (MnX) (Fig.
15D), the nucleus tractus solitarii (S)(Fig. 15D), and the
nucleus nervi hypoglossi (nXII) express R-cadherin.
R-cadherin-positive fiber fascicles were found in the
lemniscus lateralis (LL) (Fig. E A ) , the corpus trapezoi-
deum, and the brachium cerebellopetale (BCP) (Fig. 15A).
Moreover, axon fascicles of the vagus nerve (fX)(Fig.
15C,D) and ventral fibers of the acoustic nerves (fVIIIv)
(Fig. 15B)express R-cadherin.
DISCUSSION
By immunohistochemical and/or in situ hybridization
techniques, the expression of R-cadherin in the embryonic
chicken brain was studied at three intermediate stages of
development (8,11, and 15 days ofincubation; E8, E l l , and
E15). At these stages, R-cadherin is expressed in a re-
stricted fashion by particular developing gray and white
matter resons throughout the brain (Table 1). Compared
to a previous study, which focused on the tectofugal
systems and used weaker fluorochromes (Redies et al.,
19931, the number of R-cadherin-positive regions identified
in the present study is relatively large.
R-cadherin expression by particular
neural circuits
The immunostaining data show that some of the R-
cadherin-positive fiber tracts originate and terminate in
R-cadherin-positive gray matter structures. This result
indicates that some of the R-cadherin-positive neuroana-
tomic structures listed in Table 1 form parts of particular
information processing units in the brain, as has been
previously shown for N- and R-cadherin in the tectofugal
system of the chicken (Redies et al., 1993) and for N-
cadherin in the mouse (Redies and Takeichi, 1993a). We
therefore asked which functional systems and circuits the
R-cadherin-positive neuroanatomic structures belong to
and whether we can find any common denominator for
their functional specificity.
Visual system. R-cadherin is expressed by parts of the
retino-recipient and tectofugal systems. In the retino-
recipient system, R-cadherin expression is observed in the
nucleus suprarotundus and the nucleus marginalis of the
optic tract (Gunturkun and Karten, 1991). Most of the
other retino-recipient nuclei as well as the retinofugal fibers
do not express R-cadherin but rather express N-cadherin
R-CADHERIN EXPRESSION IN CHICKEN BRAIN
Fig. 10. Frontal sections of the ventral thalamus from E8 (A,B)and
E l 5 (C-F)embryos immunostained for neurofilament (A,C,E) and for
R-cadherin I B,D,F),showing the ansa lenticularis. The panel pairs A,B,
C,D, and E,F represent double-staining results from the same section,
respectively. The panels shown in E,F represent enlargements of the
(Matsunaga et al., 1988; Inuzuka et al., 1991b; Redies and
Takeichi, 1993b; Redies et al., 1993).
A schematic representation of the R-cadherin-positive
structures of the tectofugal projections is shown in Figure
387
regions indicated by the hatched boxes in C,D. Note that the neurofila-
ment and the R-cadherin staining patterns are virtually identical,
suggesting that R-cadherin is expressed by neurite fascicles. Abbrevia-
tions are listed separately. Scale bars = 100 pm.
19. These results confirm previous observations that R-
cadherin is expressed by different tectal layers, by the
tecto-isthmic projection, and by its target nuclei (i.e., the
isthmic nuclei) (Imc, Ipc, SLu, 1 0 ) (Redies et al., 1993) and
388 K. ARNDT AND C. REDIES
TeO

- IH

d
I

Irnc I

Fig. 11. Schematic representation of R-cadherin expression in
frontal sections of a n E l l mesencepahlon displayed in a rostral-to-
caudal sequence. Sections are a t levels of the nucleus ansae lenticularis
posterior ( A J , the nucleus mesencephalicus lateralis, pars dorsalis
IB-D), and of the isthmic nuclei (El, as shown in Figure 2. In each
section, the midline is on the right-hand side. An example of the
primary data is shown in Figure 1. R-cadherin expression by gray
matter regions is indicated by dotted areas. More densely dotted areas
exhibit stronger immunostaining. Black squares represent R-cadherin-
positive axon fascicles. Abbreviations are listed separately. Scale bars =
500 iJ-m.
K-CADHEKIN EXPRESSION IN CHICKEN BRAIN
..
d In the ascending auditory system, R-cadherin-positive
I and parts of the primary auditory nucleus, the nucleus
V angularis cochlearis (An),and the nucleus lemnisci latera-
Figure 11 (Continued.)
Fig. 12. Frontal section of the nucleus oculomotorius from an E l 5
embryo immunostained for R-cadherin protein. T h e arrowheads point
to perikarya of R-cadherin-expressing neurones. Note the surface
membrane staining. The midline is on the left side. Scale bar = 50 ym.
by parts of the tecto-thalamic system. N-cadherin is also
expressed by the tectofugal system in a largely complemen-
tary pattern (Redies et al., 1993). Of the thalamic nuclei
that receive direct efferents via the tecto-thalamic projec-
tions, R-cadherin is expressed by clusters of cells in the
nucleus rotundus (ROT),by parts of the nucleus subrotun-
dus tSRt), by the nucleus triangularis (T), by the stratum
cellulare externum (SCE), and by scattered cells in the
nucleus pretectalis (PT) and in the nucleus interstitio-
389
pretecto-subpretectalis (Table 1; Figs. 1, 5-7, 19). The
nucleus rotundus is the major tecto-recipient nucleus of the
thalamus processing visual information. It can be subdi-
vided into several compartments on the basis of histochemi-
cal mapping (Martinez-de-la-Torreet al., 1990),electrophysi-
ologic data (Wang et al., 1993), and efferent projections
from the optic tectum (Benowitz and Karten, 1976).
Rotunda1 projections to the telencephalon terminate in
the ectostriatum (E) (Karten and Hodos, 1970; Benowitz
and Karten, 1976; Minelli et al., 1979). This nucleus, as well
as part of the lateral forebrain bundle (FPL), which con-
nects the nucleus rotundus with the ectostriatum, are
R-cadherin positive (Fig. 19). By contrast, the main areas of
the visual wulst and its efferents (Karten et al., 1973;
Wilson, 1980; Reiner and Karten, 1983) do not express
R-cadherin.
Auditory system. The R-cadherin-positive fiber tracts
and nuclei that constitute parts of the ascending and
descending auditory system are schematically shown in
Figure 20.
structures include the entire nucleus olivaris superior (0s)
lis, pars intermedia (LLi) at the rhombencephalic level.
These structures are interconnected via the lemniscus
lateralis and the corpus trapezoideum (Boord, 1968; Conlee
and Parks, 1986), which contain R-cadherin-positive fas-
cicles at E8. Efferents of these nuclei project rostrally to the
nucleus mesencephalicus lateralis, pars dorsalis, of the
torus semicircularis (MLd) (Karten, 1967; Boord, 1968;
Conlee and Parks, 1986), which contains an R-cadherin-
positive area. Projections from this nucleus terminate
mainly in the nucleus ovoidalis (Ov) (Karten, 1967; Durand
et al., 1992) and its surrounding shell region in the dien-
cephalon (sOv) (Durand et al., 1992). Parts of both struc-
tures express R-cadherin. Ascending projections from the
ovoidal region terminate in several telencephalic regions
(Durand et al., 1992; Wild et al., 1993). These regions
include parts of the neostriatum caudale (NC), the neostria-
tal region that borders the ectostriatum, paleostriatal re-
gions, the nucleus taeniae, the tuberculum olfactorium,
ventral parts of the archistriatum, and several cell groups
of the hyperstriatum ventrale. All these structures express
R-cadherin (Table 1; Figs. 3, 4). The main telencephalic
auditory center, Field L, is R-cadherin negative.
In the descending auditory system (Fig. 201, the R-
cadherin-positive ovoidal region projects, via the lateral
forebrain bundle (FPL), to the following nuclei at the
hypothalamic level, which also express this molecule: the
nucleus inferioris hypothalami (IH),the nucleus ventrome-
dialis hypothalami (VMN), and a few cell groups in the
anterior and lateral hypothalamus (Durand et al., 1992).
Moreover, a part of the nucleus intercollicularis (for a
detailed discussion of the nucleus intercollicularis subdivi-
sions, see also Puelles et al., 1994),the nucleus dorsomedia-
lis (DM) (Wild and Arends, 1987; Deviche and Gunturkun,
1992), expresses R-cadherin in its entirety. This nucleus
receives projections from an R-cadherin-positive area in
the archistriatum and transmits information to the syrin-
geal part of the R-cadherin-positive nervus hypoglossus.
7’rigeminal system. Elements of the somatosensory
trigeminal circuit (Zeigler and Karten, 1973; Dubbeldam et
al., 1981; Berkhoudt et al., 1982; Wild et al., 1985; Schall et
al., 1986) that express R-cadherin are schematically shown
390 K. ARNDT AND C. REDIES

Fig. 13. Frontal sections of the isthmic region from E8 (A,B),E l l (C,D),and E l 5 (E,F) embryos
stained for Nissl substance (A.C,E) and immunostained for R-cadherin (B,D,F).The panel pairs A,B, C,D,
and E,F represent adjacent sections. The arrowhead in B points to neurite fascicles of the tecto-isthmic
tract (TTI1 expressing R-cadherin. Abbreviations are listed separately. Scale bars = 100 pm.

in Figure 21 and include parts of the nucleus principalis
sensorius nervi trigemini in the rhombencephon (nPrV),
parts of the nuclei associated with the tractus descendens
nervi trigemini (nTTD), cell groups of the nucleus reticu-
lark parvocellularis (Rpc),several cerebellar lobules (Figs.
16-18),and, in the telencephalon, the nucleus basalis (Bas),
parts of the neostriatum frontale (NF), and the archistria-
tum intermedium (AI) (Table 1;Fig. 3).
These structures are interconnected by fiber tracts, some
of which express R-cadherin. For example, the tractus
quintofrontalis (QF) (Wild et al., 1985; Schall et al., 1986) is
R-cadherin positive at stage E l 9 (data not shown). Further-
more, the nucleus principalis sensorius nervi trigemini
(nPrV) sends efferents to the contralateral side via R-
cadherin-positive fibers crossing at the level of the nuclei
oculomotorius et trochlearis (Dubbeldam et al., 1981), and
 R-CADHERIN EXPRESSION IN CHICKEN BRAIN
Fig. 14. Frontal sections of the isthmo-optic nucleus from E l l
!A,B)and E l 5 !C-E)embryos stained for Nissl substance !A,C) and for
cell nuclei with Hoechst 33258 dye (D) and immunostained for R-
cadherin IB,Ei. The panel pairs A,B and C,D represent adjacent
sections. The panel pair D,E represents double-staining results from
to the nucleus basalis (Bas) (Zeigler and Karten, 1973;
Dubbeldam et al., 1981; Berkhoudt et al., 1982; Wild et al.,
1985; Schall et al., 1986). The nucleus lemnisci lateralis,
pars intermedia (LLi),an auditory nucleus, also projects to
the nucleus basalis in birds (Arends and Zeigler, 1989a;
Hall et al., 1993).Efferents of the nucleus basalis terminate
in the overlying part of the neostriatum. This area, in turn,
projects to the neostriatum caudale and archistriaturn
intermedium (AI)(Berkhoudt et al., 1982; Wild et al., 1985;
Schall et al., 1986).The archistriatum intermedium projects
to the nucleus reticularis parvocellularis (Rpc),a premotor
nucleus in the rhombencephalon (Berkhoudt et al., 1982;
Wild et al., 1985; Schall et al., 1986).
The subnucleus oralis and interpolaris of the nucleus
tractus descendentis nervi trigemini (nTTD) partially ex-
press R-cadherin. Some efferents of these nuclei reach the
cerebellum (Arends et al., 1984; Arends and Zeigler, 1989b)
whereas others project to diencephalic nuclei (e.g., to the
R-cadherin-positive thalamic nucleus dorsolateralis poste-
rior, a multisensory integration center) (Gamlin and Co-
hen, 1986; Korzeniewska and Gunturkiin, 1990).
391
the same section. In each section, the midline is on the left side. The
arrowheads in D,E point to neuropil a t the outer margin of the
isthmo-optic nucleus. Abbreviations are listed separately. Scale bars =
100 +m.
Cerebellum and vestibular system. R-cadherin expres-
sion in the cerebellum is restricted to two to three parasag-
ittal zones in the lobules, to the auricle, and to the nucleus
cerebellaris lateralis (CbL) (Figs. 15A,B, 16-18).
In the adult, the cerebellar cortex is organized into
longitudinal (parasagittal)zones, which probably represent
a functional subdivision of the cerebellum (Goodman et al.,
1964; Oscarsson, 1979; Arends and Zeigler, 1991a). The
lobules of the cerebellum receive efferents from several
brainstem and thalamic regions (Karten and Finger, 1976;
Brauth, 1977; Clarke, 1977; Arends et al., 1984; Arends and
Zeigler, 1989b) and project to different cerebellar and
vestibular nuclei (Wold, 1981; Arends and Zeigler, 1991a,b;
Arends et al., 1991). The R-cadherin-positive longitudinal
zones possibly correspond to parts of zones A, C, E and F of
Arends and Zeigler (1991a). Moreover, there is a partial
overlap between the R-cadherin-positive zones and the
cerebellar termination areas of the afferents from nuclei
associated with the nucleus tractus descendentis nervi
trigemini (nTTD) (Fig. 21) in lobules V, VI, VII and,
possibly IV, in the mallard (Arends et al., 1984).
 392
Fig. 15. Schematic representation of R-cadherin expression in
frontal sections of an E l l hindbrain displayed in a rostral-to-caudal
sequence. This figure is a continuation of the results shown in Figures
3,5, and 11, but sections are cut a t a slightly different angle for a better
display of the results. Consecutive sections from the level of mid-
hindbrain to the caudal hindbrain are displayed in a rostral-to-caudal
sequence, a s shown in Figure 2. Sections are from the hindbrain a t
K. ARNDT AND C. REDIES
levels of the caudal cerebellum (A,B) and of the sensory and motor
nuclei of the vagus nerve (C,D).In each section, the midline is on the
right side. R-cadherin expression by gray matter regons is indicated by
dotted areas. More densely dotted areas exhibit stronger immunostain-
ing. Black squares represent R-cadherin-positive axon fascicles. Abbre-
viations are listed separat.ely. Scale bar = 500 pm.
R-CADHERIN EXPRESSION IN CHICKEN BRAIN
Fig. 16. Frontal sections of the cerebellum and hindbrain from an E l 1embryo stained for Nissl substance (A),R-cadherin
transcript (B),and R-cadherin protein tC). The three panels represent consecutive sections. The midline is on the right side.
Abbreviations are listed separately. Scale bar = 500 bm.
Zones E and F project to vestibular nuclei including
R-cadherin-positive areas h e . , cell groups in the nuclei
vestibulares descendens et medialis). The efferents of zone
C terminate in the R-cadherin-positive nucleus cerebellaris
lateralis (CbL) (Arends and Zeigler, 1991a). The targets of
the projections arising from the nucleus cerebellaris latera-
lis include R-cadherin-positive parts of the vestibular nu-
clei and the thalamic nucleus dorsolateralis intermedius
(DLI) (Fig. 23) (Arends and Zeigler, 1991b). Projections
from the R-cadherin-positive vestibular nuclei, in turn,
terminate in the nucleus oculomotorius, including the
nucleus of Edinger-Westphal (Wold, 1981; Arends et al.,
1991),which also express this cadherin.
Motor system. Other examples of functional circuits
that consist of R-cadherin-expressing nuclei and fiber
tracts are found in the motor system (Figs. 22, 23). An
important telencephalic source of descending motor control
is the paleostriatal complex, which consists of a magnocellu-
lar subdivision (i.e., the paleostriatum primitiwm I PPI and
the nucleus intrapeduncularis [INPI), and a parvocellular
subdivision (i.e., the paleostriatum augmentatum [PA], the
lobus parolfactorius [LPO1, and the nucleus accumbens)
(Karten and Dubbeldam, 1973; Kitt and Brauth, 1981).
These structures partially express R-cadherin. The paleo-
393
striatum primitivum (PP)projects via the ansa lenticularis
(AL) to the followingdi- and mesencephalic nuclei (Fig. 22):
the nuclei ansae lenticularis anterior et posterior (ALa,
ALp), the nucleus spiriformis lateralis (SpL), the nucleus
pretectalis (PT), and the substantia nigra, and pars com-
pacts (SNc)(Karten and Dubbeldam, 1973; Kitt and Brauth,
1981; Reiner et al., 1982; Reiner et al., 1983; Veenman and
Reiner, 1994; Veenman et al., 1994). All these structures
express R-cadherin.
Afferents to the paleostriatal complex (Fig. 23) originate
in nuclei of the di-, mes-, and rhombencephalon (Kitt and
Brauth, 1982; Kitt and Brauth, 1986a,b; Wild, 1987). Some
of these structures partially express R-cadherin (e.g., the
locus coeruleus and nucleus subcoeruleus I LoC, SC], the
substantia nigra, pars compacta [SNc], and the nucleus
dorsolateralis intermedius thalami [DLII). Parts of the
cerebellum and some of the nuclei receiving cerebellar
projections also express R-cadherin. One of these nuclei, the
nucleus cerebellaris lateralis, projects to the nucleus dorso-
lateralis intermedius thalami (Arends and Zeigler, 1991b).
Conclusion. The above examples suggest that the expres-
sion of R-cadherin by brain nuclei and fiber tracts is not
random but linked to particular neural circuits within
several functional systems. Although there seems to be no
394
Fig. 17. Transverse sections of the cerebellum from a n Ell embryo
stained for Nissl substance ( A )and for cell nuclei with Hoechst 33258
dye ( C ) and immunostained for R-cadherin (B,D). Panel pair C,D
represents double-staining results from the same section and shows an
obvious common denominator for the function of all R-
cadherin-positive structures, the overall predominance of
structures involved in the control of motor functions,
including those exhibiting convergence of multisensory
inputs, is noteworthy. In the spinal cord of the chicken,
R-cadherin is expressed by the ventral motor column
(Inuzuka et al., 1991b). In conclusion, R-cadherin belongs
to a class of molecules that are markers for particular
functional circuits in the brain. Other members of this class
include Cat-301 (Hockfield et al., 1983; Hockfield et al.,
1990), TAG-liaxonin-1 (Yamamoto et al., 1986; Wolfer et
al., 1994), and N-cadherin (Redies et al., 1992; Redies and
Takeichi, 1993a; Redies et al., 1993).
K. ARNDT AND C. REDIES
enlargement of the region indicated by the hatched box in B. Panel pair
A,B represents adjacent sections. The arrowheads in C,D point to the
outer limit of the molecular layer. Abbreviations are listed separately.
Scale bars = 250 km in A and B, 50 p m in C and D.
R-cadherin expression and the formation
of nuclei
The present results extend our previous observations on
the restricted expression of R-cadherin in parts of particu-
lar neuromeres at earlier stages of chicken brain develop-
ment (E3-E6) (Ganzler and Redies, 1995). During this
period, R-cadherin-positive neurons begin to accumulate in
the mantle layer and aggregate into specific (pro-)nuclei.At
the ensuing stages of brain development examined in the
present study (E8, E l l , and E15), the expression of R-
cadherin closely parallels the cyto-architectonicdifferentia-
tion of brain gray matter. At E8, when the boundaries
 A
B d -
V
Fig. 18. Display of R-cadherin immunostaining in the cerebellum of
an Ell embryo. A: Sagittal view of the cerebellum with the developing
lobules indicated by Roman numerals. The arrows indicate the orienta-
tion of view for the irnmunostaining results displayed (B).The immuno-
staining results were reconstructed from a series of consecutive trans-
Hb
verse sections spaced 20 p.m apart. The thin lines represent strong
immunostaining extending from the outer limit of the molecular layer
to the internal granular layer (see Figure 17).The staining pattern was
transferred onto an outline of the cerebellum and hindbrain. Abbrevia-
tions are listed separately. Scale bar = 80 ym.
396
Fig. 19. Schematic representation of the R-cadherin-positive neuro-
anatomic structures in the visual (tectofugal) system of the embryonic
chicken brain. The dotted areas represent gray matter regions express-
ing R-cadherin. The thick arrows represent R-cadherin-positive fiber
tracts. The thin arrows represent some of the known fiber tracts
connecting the R-cadherin-positive gray matter areas (see Discussion);
these fiber tracts could not be identified on the sections or they did not
express R-cadherin. Abbreviations are listed separately.
V Fig. 22. Schematic representation of the R-cadherin-positive effer-
Fig. 20. Schematic representation of the R-cadherin-positive neuro-
anatomic structures in the auditory system of the embryonic chicken
brain. Symbols are explained in the legends to Figure 19. Abbreviations
are listed separately.
between individual nuclei are still relatively ill defined in
most brain regions, R-cadherin expression in the develop-
ing mantle layer is restricted to particular regions; many of
these regons show a gradual decrease of R-cadherin expres-
sion at their borders. As the architecture of the various
brain areas matures and nuclei become morphologically
more distinct between E8 and E15, the borders of R-
cadherin expression sharpen and outline particular nuclei
or regons within nuclei (Figs. 6, 7, 13, 14).
In some gray matter structures (e.g., the nucleus rotun-
dus, the nucleus pretectalis, and the paleostriatum augmen-
tatum ), R-cadherin expression becomes restricted to clus-
ters of cells or to a population of dispersed cells (Figs. 4,
6-8). The R-cadherin-positive cells have elaborate pro-
cesses and relatively large nuclei, suggesting that they are
neurons (Peters et al., 1970).The morphology of individual
K. ARNDT AND C. REDIES
Fig. 21. Schematic representation of the R-cadherin-positive neuro-
anatomic structures in the trigeminal system of the embryonic chicken
brain. Symbols are explained in the legends to Figure 19. Abbreviations
are listed separately.
ents from the paleostriatal complex of the embryonic chicken brain.
Symbols are explained in the legends to Figure 19. Abbreviations are
listed separately.
R-cadherin-positive cells is not always clear from the
immunostaining results, perhaps because R-cadherin is a
glycoprotein expressed on the surface of the cells and their
processes. In many brain structures, the R-cadherin-
positive processes seem to form interwoven networks,
thereby obscuring the morphology of individual R-cadherin-
expressing cells (Figs. 7C,D, 8C,D).
A comparison of immunohistochemical and in situ hybrid-
ization results shows that almost all gray matter structures
that were found to be R-cadherin positive by immunostain-
ing also express R-cadherin mRNA (Figs. 4, 7, 8, 16).
Obvious regional mismatches between results obtained
with the two techniques were only observed in the hippocam-
pus, which expresses R-cadherin transcript but shows weak
immunostaining signal. However, on the single-cell level,
mismatches cannot be excluded because we did not carry
out double-label staining on the same sections. Moreover, it
 R-CADHERIN EXPRESSION IN CHICKEN BRAIN
V former cells is too low to permit them encountering each
Fig. 23. Schematic representation of t h e R-cadherin-positive affer-
ents to the paleostriatal complex of the embryonic chicken brain.
Symbols are explained in the legends to Figure 19. Abbreviations are
listed separately.
is not possible to clearly correlate cellular processes visual-
ized by immunostaining with perikarya expressing R-
cadherin mRNA.
Much neuronal differentiation occurs between E8 and
E l 1in areas such as the forebrain, and we did not follow the
morphologic changes during this developmental period in
detail in this study, except for the nucleus rotundus (Fig. 6).
Despite this limitation, the present results support the
idea that cadherins play a morphogenetic role in the early
development of brain nuclei (Redies et al., 1993; Ganzler
and Redies, 1995; Redies, 1995). Cadherins mediate homo-
typic cell-cell adhesion, as demonstrated by numerous in
vitro experiments showing that cells expressing the same
type of cadherin selectively aggregate and segregate from
cells expressing other cadherins. This selective adhesive-
ness is thought to be based, at least in part, on the
preferentially homophilic binding between cadherin mol-
ecules (reviewed in Ranscht, 1991; Takeichi, 1991; Geiger
and Ayalon, 1992; Kemler, 1993; Redies, 1995; Takeichi,
1995). Heterophilic binding between different types of
cadherins has also been described, but it is generally weaker
than homophilic binding (Inuzuka et al., 1991a; Matsunami
et al., 1993; Nakagawa and Takeichi, 1995).
The formation of different types of tissue architecture
from differentially adhesive cells can be predicted by cell
aggregation models that take into account the density of the
different cell populations and their specific binding affini-
ties (reviewed in Steinberg, 1963; Steinberg and Poole,
1981; Glazier and Weaire, 1992; Graner, 1993; Graner and
Sawada, 1993). We have recently proposed an extension of
these ideas based on our studies of cadherins to account for
the formation of gray matter architecture in the vertebrate
brain (Redies, 1995).In the present study, we observed that
the R-cadherin-expressing cells form the same basic types
of tissue architectures that were predicted by the cell
aggregation models:
1.The two populations may completely segregate into cell
aggregates containing only one type of cell (homotypic
aggregates). Examples for largely homotypic aggregates of
R-cadherin-expressing cells are the nucleus isthmi (Fig.
13), the nucleus spiriformis lateralis, and the neostriatum
caudale (Fig. 1).
397
2. The two populations may incompletely segregate, with
one cell type surrounding an aggregate of the other cell type
like a shell. A shell-like aggregation of R-cadherin-positive
cells around another nucleus is, for example, the nucleus
marginalis tractus optici, which surrounds the nucleus
rotundus (Figs. 1,5B,C, 7 A - 0 , and the shell nucleus of the
nucleus ovoidalis (Figs. 1,5B).
3. The two populations may form small aggregates of one
cell type within a larger aggregate of cells of the other type.
Smaller clusters of R-cadherin-positive cells are seen, for
example, in the nucleus rotundus (Figs. 1,5B, 6, 7), in the
nucleus intercollicularis (Fig. 11B,C), and in the hypotha-
lamic gray matter (Figs. 3E,F, 5A-D, 11A-C).
4.Finally, cells of one type may remain dispersed within a
large aggregate of cells of another type if the density of the
other frequently within the aggregate (Steinberg, 1963).
Examples are the R-cadherin-expressing cells of the nucleus
pretectalis (Fig. 8 ) and of the nucleus interstitio-pretecto-
subpretectalis. In these nuclei, the R-cadherin-positive
cells are dispersed within large numbers of N-cadherin-
positive cells (Redies et al., 1993). N- and R-cadherin can
bind heterophilically to each other (Inuzuka et a]., 1991a;
Matsunami et a]., 1993).
In conclusion, the pattern of expression observed in this
study suggests that R-cadherin plays a role in the formation
and differentiation of specific gray matter structures in the
chicken brain. It would be of interest to study whether, in
general, all gray matter structures express a particular
cadherin (or a combination of cadherins). Partially comple-
mentary expression patterns have been demonstrated for
R- and N-cadherin in the central nervous system of the
chicken embryo (Inuzuka et al., 1991a; Inuzuka et al.,
1991b; Redies et al., 1992; Redies et al., 1993; Ganzler and
Redies, 1995). Other cadherins expressed in the vertebrate
brain are also differentially expressed in developing gray
matter areas (Shimamura and Takeichi, 1992).
R-cadherin expression and the formation
of fiber tracts
This study shows that, throughout the embryonic chicken
brain, R-cadherin is selectively expressed in particular fiber
tracts (Table 1; Figs. 9, 10).The most prominent staining is
observed in large tracts composed of prominent fiber
bundles. Dispersed R-cadherin-positive fiber bundles were
not found, perhaps due to the relatively weak staining of
neurites at the stages examined in this study.
Most, if not all, of the R-cadherin-positive fiber tracts
(e.g. the stria medullaris [Fig. 91, the lateral forebrain
bundle ]Fig. 101, and the decussatio supraoptica) can be
divided into R-cadherin-positive and R-cadherin-negative
fascicles. Similar observations were made for N-cadherin in
the chicken tectofugal system (Redies et al., 1993). The
expression of the two cadherins is often partially complemen-
tary within the same fiber tracts (data not shown), suggest-
ing that the two molecules may be involved in the segrega-
tion of fiber bundles or in their selective fasciculation (see
also Redies et a]., 1992; Redies et al., 1993).
Because R-cadherin can induce neurite elongation (Redies
and Takeichi, 1993b)and is selectively expressed by particu-
lar fiber tracts or parts of fiber tracts, it is conceivable that
R-cadherin acts as a guidance cue for axon navigation, as
has been suggested for N-cadherin (Matsunaga eta]., 1988;
398
Redies et al., 1992; Redies et al., 1993) and T-cadherin
(Fredette and Ranscht, 1994).The expression of R-cadherin
during the time of axon elongation (about E3-Ell) and its
later down-regulation is compatible with this suggestion.
General conclusion
Cadherins are morphogenetic molecules regulating the
aggregation and sorting of neural cells and the outgrowth
and fasciculation of neurites (reviewed in Takeichi et al.,
1990; Ranscht, 1991; Dalseg et al., 1993; Redies, 1995).
Results from this study suggest that their expression
during brain development may be directly involved in the
formation of particular brain structures such as brain
nuclei, cortical layers, and fiber tracts and, finally, in the
establishment of neural circuitry. The specific functions of
cadherins and other adhesion molecules in these develop-
mental processes remain to be experimentally established.
ACKNOWLEDGMENTS
We are grateful to 0. Gunturkun for neuroanatomic
advice, to M. Takeichi for R-cadherin cDNA, to H. Fujisawa
for neurofilament antibody, to U. Schwarz for generous
support of this study, to Vicky Kastner for technical
assistance and English corrections, to S. Ganzler and K.
Korematsu for discussion and comments on the manu-
script, and to two anonymous reviewers for constructive
criticism. This work was supported by the Max Planck
Society.
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