/. Embryol. exp. Morph. Vol. 41, pp. 223-232, 1977
Printed in Great Britain © Company of Biologists Limited 1977
223
Ultrastructural analysis of the apical ectodermal
ridge during vertebrate limb morphogenesis
II. Gap junctions as distinctive ridge structures common
to birds and mammals
By JOHN F. FALLON 1 AND ROBERT O. KELLEY 2
From the Department of Anatomy, The University of Wisconsin
and Department of Anatomy, The University of
New Mexico
SUMMARY
The fine structure of the apical ectodermal ridge of five phylogenetically divergent orders
of mammals and two orders of birds was examined using transmission and freeze fracture
electron microscopy. Numerous large gap junctions were found in all apical ectodermal
ridges studied. This was in contrast to the dorsal and ventral limb ectoderms where gap
junctions were always very small and sparsely distributed. Thus, gap junctions distinguish
the inductively active apical epithelium from the adjacent dorsal and ventral ectoderms.
The distribution of gap junctions in the ridge was different between birds and mammals
but characteristic within the two classes. Birds, with a pseudostratified columnar apical ridge,
had the heaviest concentration of gap junctions at the base of each ridge cell close to the
point where contact was made with the basal lamina. Whereas mammals, with a stratified
cuboidal to squamous apical ridge, had a more uniform distribution of gap junctions throughout the apical epithelium. The difference in distribution for each class may reflect structural
requirements for coupling of cells in the entire ridge. We propose that all cells of the apical
ridges of birds and mammals are electrotonically and/or metabolically coupled and that this
may be a requirement for the integrated function of the ridge during limb morphogenesis.
INTRODUCTION
The limb-buds of birds and mammals are capped apically by a thickened
epithelium, termed the apical ectodermal ridge, which is required for normal
limb development (Saunders, 1948; Zwilling, 1961). Should the ridge be removed
surgically (Saunders, 1948) or disappear because of mutation (Milaire, 1965),
limb development will stop at the time the ridge is absent. Thus the apical
ridge is considered an inducer of limb development.
In a previous investigation (Kelley & Fallon, 1976), we have reported that
the apical ectodermal ridge of the human embryonic limb contained large gap
1
Author's address: Department of Anatomy, 453 Bardeen Medical Laboratories, The
University of Wisconsin, Madison, WI 53706, U.S.A.
2
Author's address: Department of Anatomy, The University of New Mexico, School of
Medicine, Albuquerque, NM 87131, U.S.A.
15-2
224
J. F. FALLON AND R. O. KELLEY
junctions between ridge cells. The size and distribution of these junctions contrasted sharply with our observation that gap junctions between cells of dorsal
and ventral ectoderm were sparsely distributed and/or small if present at all.
Although apical ridges of several other mammalian and avian species have been
examined with the electron microscope (e.g. Jurand, 1965; Ede, Bellairs &
Bancroft, 1974; Schweichel, 1972) none reported gap junctions except Gould,
Day & Wolpert (1972). These investigators in reporting on cell contacts in early
chick wing morphogenesis noted in passing, considerable numbers of gap junctions
in thin sections of the chick apical ridge. However, no comparison was made
with dorsal and ventral ectoderms to establish whether or not large and extensive gap junctions were distinctive features of the apical ectodermal ridge.
We have examined the fine structure of the ridge of five orders of phylogenetically divergent mammals and two orders of birds. All species examined
had in common numerous, large gap junctions between cells of the apical ectodermal ridge. However, the distribution of these gap junctions was different
between the two classes. Finally, gap junctions were infrequent and of small
dimension in both dorsal and ventral limb ectoderms in all species.
MATERIALS AND METHODS
Intact limb-buds were dissected from the avian and mammalian embryos
listed in Table 1 and selected for development comparable to stage 22-24 of the
chick embryo limb-bud (Hamburger & Hamilton, 1951) and stage 14-15 of
human limb morphogenesis (O'Rahilly, Gardner & Gray, 1956).
For transmission electron microscopy, all avian and mammalian specimens
were fixed for 2 h at 4 °C by immersion in a modified Bouin's fixative containing
0-02 % trinitrophenol, 2-0 % formaldehyde (Stefanini, Martino & Zamboni,
1967) and 2-5 % glutaraldehyde (Ito & Karnovsky, 1968) buffered to pH 7-2
with either 0-075 M phosphate (avian) or 0-1 M cacodylate (mammalian) buffer.
Specimens were rinsed in appropriate buffer and postfixed for 2 h (1 h on ice;
1 h at room temperature) in 1*0 % osmium tetroxide buffered to pH 7-4 with
0-1 M s-collidine buffer. Thin sections were stained for lOmin in ethanolic
uranyl acetate and for 3 min in alkaline lead citrate prior to examination.
For freeze fracturing, limb-buds were prepared as described in Kelley and
Fallon (1976). In this report the terms P-face (formerly A) and E-face (formerly
B) particles indicate those intramembrane subunits which remain associated with
inner and outer portions, respectively, of the fractured cell membrane. Illustrations of replicas are presented with the platinum shadow direction approximately from the bottom to the top of the plate.
Gap junctions in ectodermal ridges of birds and mammals
225
Table 1. Animals examined for gap junctions in limb-bud epithelia
Common name
Swiss mouse
Guinea pig
Hamster
Rabbit
Ferret
Pig
Horse
Genus
M'us
Cavia
Mesocricetus
Oryctolagus
Mustelo
Sus
Equus
Class Mammalia
Family
Species
musculus
Muridae
porcillus
Caviidae
auratus
Cricetidae
cuniculus
Leporidae
putorius
Mustelidae
scrofa
Suidae
cabal 1 us
Equidae
Order
Rodentia
Rodentia
Rodentia
Lagomorpha
Carnivora
Artiodactyla
Perissodactyla
Common Name
Chicken
Quail
Pheasant
Pea fowl
Chuckar
partridge
Turkey
Guinea fowl
Muscovey duck
Genus
Gall us
Coturnix
Phasianis
Pavo
Class Aves
Species
domesticus
coturnix japonica
colchicus
cristatus
Family
Phasianidae
Phasianidae
Phasianidae
Phasianidae
Order
Galliformes
Gal li formes
Galliformes
Galliformes
Alectoris
Meleagris
Numida
Cairina
graeca
gallopavo
meleagris
moschata
Phasianidae
Meleagrididae
Numididae
Anatidae
Galliformes
Galliformes
Galliformes
Anseriformes
OBSERVATIONS
A list of species examined in this investigation is presented in Table 1. Large
well-developed gap junctions were observed in the apical ridge of each species
in marked contrast to smaller, more sparsely distributed gap junctions in
adjacent non-ridge ectoderm. Micrographs from each species listed in Table 1
are not illustrated. However, because the distribution of gap junctions was
similar according to class (Aves versus Mammalia), representatives from each
class are shown.
Organization of gap junctions in the apical ridge of birds
A transverse section through the wing bud of a stage-22-24 chick (Fig. 1)
exhibits a thickened, pseudostratified columnar epithelium composed of closely
aggregated cells and distal periderm. Nuclei are euchromatic and organized
in multiple layers throughout the ridge. Examination of thin sections of the
ridge reveals numerous gap junctions, approximately 0-75 to 1-20 [im in length,
at apposed lateral surfaces (arrows, Fig. 2). Junctions are most numerous on
lateral borders of cells toward the basal lamina. Frequently a single cell is
coupled to several contiguous cells. Higher magnification of the basal region
of the ridge (Fig. 3) reveals gap junctions of several dimensions. Profiles of the
larger structures (large arrows) approach 2-0 ^m in length. Fig. 3 also shows a
portion of one cell (number 1) which has developed gap junctions with at least
226
J. F. FALLON AND R. O. KELLEY
Gap junctions in ectodermal ridges of birds and mammals
227
three adjacent cells (numbers 2, 3 and 4). In addition, cell number 2 is in junctional contact with cell number 5, thus forming an apparent intercellular network of communicating junctions. Fewer and smaller junctions were seen in
more distal portions of the apical ridge. No junctions were observed between ridge
cells and the periderm. It is probable that all cells of the apical ridge are interconnected by gap junctions, although present techniques do not permit conclusive analysis of their complete distribution. In addition, an annular gap
junction was present in the cytoplasm of cell number 2.
The dorsal and ventral ectoderms were examined carefully. Gap junctions
were rarely seen. In order to emphasize this point we note that for some time we
thought that dorsal and ventral ectoderms lacked gap junctions. In fact, we
previously reported not observing them in dorsal and ventral ectoderms of
human limb-buds (Kelley & Fallon, 1976). When gap junctions finally were
observed in avian dorsal and ventral ectoderms they measured 0-2 ^m or less
in diameter. Thus, the dorsal and ventral ectoderms do develop gap junctions
but they are small in size and sparsely distributed.
Replicas of chick limb-buds (stage 22-24) freeze-fractured through the apical
ridge permit examination of the dimension of gap junctions viewed en-face.
Fig. 4 illustrates the lateral surface of a ridge cell in contact with an adjacent
cell. The fracture plane reveals P-face particles that are approximately 8-5-9-0
nm in diameter.
A similar pattern of particle aggregation is present between apposed cells in
the apical ridge of quail embryos. Fig. 5 reveals that P-face particles are organized into smaller aggregates separated by narrow, discontinuous channels. The
channels are also reflected in the E-face of the apposed cell membrane and are
evident in E-face surfaces of the junction illustrated.
Gap junctions in the apical ridge of mammals
In contrast to the pseudostratified columnar organization of the ridge in
birds, the mammalian ridge comparable to stage 15 of human limb development
FIGURES
1-3
Fig. 1. Transverse section of chick wing-bud (stage 23). Micrograph oriented with
epithelium above embryonic limb mesenchyme. Note thin periderm and pseudostratified columnar features of subjacent epithelium, x 600.
Fig. 2. Ridge cells of chick wing-bud connected by gap junctions (arrows) at
apposed lateral cell surfaces. n, nucleus; tn, mitochondrion; cm, cell membrane,
x 19000.
Fig. 3. Higher magnification of basal surface of ridge abutting basal lamina.
Profiles of larger junctions (large arrows) approach 2.0 /tm in length whereas
smaller junctions (small arrows) are only 0.05 /on in length. The latter may be
small gap junctions, or sections through the periphery of larger ones. Cell number 1 is
coupled with cells 2, 3 and 4. Note also that cell 2 is in junctional contact with cell 5.
An annular gap junction (qgj) is present in cell 2. It is probable that all cells of the
apical ridge are connected by gap junctions x 33000.
228
J. F. FALLON AND R. O. KELLEY
Gap junctions in ectodermal ridges of birds and mammals
229
is a stratified cuboidal to squamous epithelium (Fig. 6), extending the length
of the apical, antero-posterior axis of the limb-bud. Similar to the pattern observed in birds, lateral borders of cells in the mammalian ridge are extensively
connected by large gap junctions. However, these large gap junctions are distributed throughout the mammalian ridge as opposed to the more basal location in
avian ridges. Fig. 7 illustrates portions of ridge cells in a horse embryo of which
at least four cells are interconnected by prominent junctions (arrows). As in the
avian ridge, it is also probable that all ridge cells of mammalian embryos are
connected by gap junctions. In contrast to the apical ridge epithelium, the cells
of the dorsal and ventral ectoderms of the mammalian limb-buds examined
have only very small (0-2 /tm or less in diameter) and sparsely distributed gap
junctions.
Further confirmation of the identity of mammalian cell membrane specializations as gap junctions is provided by a replica of a mouse limb-bud in Fig. 8.
Similar to avian gap junctions (cf. Figs. 4 and 5), P-face particles in mouse
ridge cells are organized into smaller aggregates separated by narrow channels.
DISCUSSION
In this paper we report the presence of numerous gap junctions of large
diameter in the inductively active apical ectodermal ridge of five orders of
phylogenetically divergent mammals and two orders of birds. In each case for
this analysis, we studied the stage of development when the apical ridge was
at its greatest height. The size and distribution of gap junctions in each ridge
contrasts sharply with the sparse distribution and small diameter of gap junctions in dorsal and ventral limb ectoderm of all species examined. Our observations (unpublished) indicate that gap junctions increase in both peripheral
dimension and quantity in the avian and mammalian ridge during the period
when epithelial-mesenchymal interactions essential to normal morphogenesis
occur.
It should be emphasized that avian and mammalian apical ridge epithelia are
of two different types; pseud ostratified columnar in birds, and stratified cuboidal
to squamous in mammals. This difference between classes is further expressed in
the distribution of gap junctions within the ridge. All cells of the avian ridge
contact the basal lamina and the heaviest concentration of gap junctions is at
FIGURES
4-5
Fig. 4. Freeze-fracture replica of the P- and E-faces of apposed ridge cells in the
chick wing-bud (stage 23). A gap junction (gj), 1-0 /«n in length and 0-5 /*m in width,
exhibits P-face particles approximately 8-5-90 nm in diameter, x 38000.
Fig. 5. Freeze-fracture replicas of P- and E-faces of ridge cell membranes in a quail
wing-bud (comparable to stage 23) in the chick. Note P-face particles are organized
into smaller aggregates separated by narrow discontinuous channels (arrows).
Channels are reflected in the E-face of the apposed cell membrane, x 74000.
J. F. FALLON AND R. O. KELLEY
Gap junctions in ectodermal ridges of birds and mammals
231
the base of each ridge cell, whereas fewer junctions develop near the apex of
the ridge. In contrast, not all cells of the mammalian ridge contact the basal
lamina and the mammalian ridge has a more uniform distribution of gap
junctions throughout. These class specific distributions may reflect structural
requirements for coupling among ridge cells. In this context, we propose that
all cells within an avian or mammalian apical ectodermal ridge are electronically and metabolically coupled. In addition, we believe that this may be a
requirement for the integrated functions of the ridge during limb morphogenesis.
In summary, gap junctions have now been demonstrated in limb-buds of
six phylogenetically divergent orders (McLaughlin &Dayhoff, 1972) of mammals
(Kelley & Fallon, 1976 and this report). Similarly, gap junctions were demonstrated in seven species of the Galliformes and one species of the Anseriformes.
We conclude that gap junctions are distinctive membrane specializations in the
apical ectodermal ridge of limb-buds in the Mammalia and suggest that this
generalization may be extended throughout the class Aves.
This investigation was supported by NIH Grants HDO7402 and AG00191; by NSF grant
nos. GB2704 and GB40506; by the Graduate School Research Committee of the University
of Wisconsin; by General Research Support funds to the University of Wisconsin Medical
School and the University of New Mexico School of Medicine from the National Institutes
of Health; and by an institutional grant to the University of Wisconsin from the American
Cancer Society.
We are grateful to Drs A. W. Clark, A. J. Ladman, B. H. Lipton, H. W. Mossman,
D. B. Slautterback and K. G. Vogel, Ms M. Gibson and Ms J. Frederick for their constructive criticism of this manuscript; and to Mrs B. Kay Kirk and Mrs Raana Beckwith for technical assistance and Mrs Debra Reierson and Ms Anita Kimbrell for typing the manuscript.
We wish to thank Drs R. Auerbach, J. Beery, A. Blazkovec, W. McGibbon, M. Orsini,
and R. Shackleford for supplying us with embryonic material used in these studies.
R.O.K. is the recipient of a Research Career Development Award from the NIH (HD70407).
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Fig. 6. Transverse thick section of the apical ridge of a mouse embryo (comparable
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(Received 17 February 1977, revised 12 April 1977)