/. Embryol. exp. Morph. Vol. 46, pp. 99-1 JO, 1978
Printed in Great Britain © Company of Biologists Limited 1978
99
Identification and distribution of gap junctions in
the mesoderm of the developing chick limb bud
ROBERT O. KELLEY 1 AND JOHN F. FALLON 2
From the Department of Anatomy, The University of New Mexico and
Department of Anatomy, University of Wisconsin
SUMMARY
Sub-ridge, core, anterior and posterior borders of mesoderm were dissected from stages 2224 chick wing buds to investigate whether structures for intercellular coupling develop between
mesenchymal cells. Fine structure was examined using techniques of transmission electron
microscopy, freeze-fracture and scanning electron microscopy. Gap (communicating) junctions
which were observed between mesenchymal cells of all limb bud regions were distributed
between apposed cell bodies, points of contact between cell processes and other cell bodies,
and between contacting tips of slender cell projections. In addition, particularly in the subridge region, filopodia were observed to extend through the intercellular matrix to contact
other cells several micrometers distant.
The observations reported in this paper show that mesodermal cells throughout the limb
have the structural capability for electrotonic and metabolic coupling during a critical period
of morphogensisis in the avian limb. Whether intercellular signals which are thought to be
transmitted through gap junctions are active in normal limb development remains to be
investigated.
INTRODUCTION
During chick limb development there is a necessary and reciprocal relationship between the apical ectodermal ridge and subjacent mesoderm (Zwilling,
1955). Removal of the ridge results in the death of sub-ridge mesodermal cells
(Cairns, 1969; Janners & Searls, 1971) and further, the determination of distal
limb parts ceases (Saunders, 1942; Summerbell, 1974). In addition, placing
non-limb mesoderm beneath the ridge causes cells of the ridge to degenerate
with the subsequent disappearance of the ridge (Searls & Zwilling, 1964). These
and other observations (Zwilling, 1961) have led to the conclusion that without
the apical ridge, cells of the sub-ridge mesoderm will die. In turn, sub-ridge limb
mesoderm is required to 'maintain' the apical ridge. Furthermore, sub-ridge
mesoderm is thought to be more embryonic than the proximal limb mesoderm
(Searls, 1965). Finally, the sub-ridge mesoderm has been proposed as a 'progress zone' where positional information is specified for pattern formation
1
Author's address: Department of Anatomy, The University of New Mexico, School of
Medicine, Albuquerque, New Mexico 87131.
2
Author's address: Department of Anatomy, University of Wisconsin, School of Medicine,
Madison, Wisconsin 53706, U.S.A.
100
R. O. KELLEY AND J. F. FALLON
along the proximo-distal axis of the developing limb (Summerbell, Lewis &
Wolpert, 1973).
Intercellular communication may be important in the determination of
pattern during development (for example, the establishment of retinal polarity;
Dixon & Cronly-Dillon, 1974; Hayes, 1976). It is now well established that the
presence of gap junctions is coincident with lowered electrical resistance between
cells, as well as with the flow of small molecules from cell to cell (for review, see
Bennett, 1973). It is also clear that communication between cells some distance
from each other is mediated by gap junctions between the cells (Sheridan, 1962).
Furthermore, Furshpan & Potter (1962) were among the first to suggest that
gap junctions are the structures which facilitate transmission of information
from one cell to another during pattern specification in development.
Fundamental to the problem of spatial patterning within limb mesoderm
during morphogenesis is the elucidation of structural mechanisms which mediate
intercellular communication between mesenchymal cells of the limb. Membrane
specializations identified as gap junctions were noted by Gould, Day & Wolpert
(1978) and Gould, Selwood, Day & Wolpert (1974) in thin sections of early
chick limb cartilage (see also Searls, Hilfer & Mirow, 1972). However, unequivocal demonstration of these structures as gap junctions by freeze-fracture
techniques was not reported. In addition, their distribution throughout the limb
mesoderm was not investigated. Hence, it is important to determine whether
junctions which are capable of mediating intercellular communication (see
Staehelin, 1974, for review) do in fact develop between cells of limb mesoderm.
Furthermore, it is reasonable to inquire whether cells of the mesoderm exhibit
differences in membrane structure which indicate the potential for electrotonic
and/or metabolic coupling either in specific areas or throughout the mesoderm.
To this end, we have examined the cell membranes of chick wing mesenchyme
during stages 22-24 using techniques of freeze-fracture, transmission and scanning electron microscopy and in this paper report the presence of gap (communicating) junctions throughout the mesoderm of the limb bud.
MATERIALS AND METHODS
Fertile White Leghorn chicken eggs (46-53 g per egg; Sunnyside Hatchery,
Oregon, Wisconsin) were incubated at 32 °C for 3-5 days in a humidified
incubator and windowed according to the techniques of Z willing (1959). For
transmission electron microscopy, wing buds (stages 22-24, Hamburger &
Hamilton, 1951) were dissected and fixed for 2 h at 4 °C by immersion in
a modified Bouin's fixative containing 0-02 % trinitrophenol, 2-0 % formaldehyde and 3-0 % glutaraldehyde buffered to pH 7-2 with 0-075 M phosphate
buffer. Specimens were washed in buffer, postfixed for 2 h on ice in. 2-0 %
osmium tetroxide buffered to pH 7-4 with 0-1 M s-collidine buffer, stained en
bloc with 0-5 % aqueous uranyl acetate for 30 min (Dym & Fawcett, 1970)
Gap junctions in limb-bud mesoderm
101
Anterior
border mesoderm
Core mesoderm
Sub- ridge
mesoderm
Posterior border mesoderm
Fig. 1. Schematic diagram illustrating locations of mesoderm dissected from stage22-24 wing buds. Tissue blocks, approximately 0-3-0-5 mm3, were removed from
(1) sub-ridge mesoderm; (2) mesoderm of the anterior border; (3) core mesoderm
and (4) mesoderm of the posterior border.
rapidly dehydrated through a graded ethanol series and embedded in Epon 218.
Thin sections were stained for 10 min in ethanolic uranyl acetate and for 3 min
in alkaline lead citrate prior to examination. For scanning electron microscopy,
fixed, ethanol-substituted specimens were dried by the critical point method,
blunt dissected, mounted on aluminum stubs with silver paint, coated with an
evaporated layer of gold-palladium alloy and examined in an ETEC Autoscan
scanning electron microscope.
For freeze-fracturing, wing buds were removed from embryos, placed in
Ham's F10 tissue culture medium and further dissected into four pieces: (1)
mesoderm immediately subjacent to the apical ridge; (2) mesoderm of the
anterior border: (3) core mesoderm; and (4) mesoderm of the posterior border,
each about 0-3-0-5 mm3 (Fig. 1). Specimens were immersed for 30 min in 2-0 %
glutaraldehyde buffered to pH 7-4 with 0-1 M cacodylate buffer (pH 7-4; 2 h;
room temperature). Some specimens were fixed prior to dissection. No differences were observed between the two methods of preparation. Tissues were
placed on gold stubs, rapidly frozen in the liquid phase of partially solidified
Freon 22 (monochlorodjfluoromethane), and stored in liquid nitrogen. Frozen
specimens, fractured at —115 °C, were replicated without etching in a Balzers
apparatus (Balzers High Vacuum Corporation, Santa Ana, California). Replicas,
cleaned with methanol and bleach, were then mounted on uncoated grids. Thin
sections and replicas were examined in either an Hitachi HU-11C, AEI 801 or
Philips 200 electron microscope.
For semi-quantitative analysis of freeze-fractured specimens, a minimum of
seven replicas were made from each area: sub-ridge; core; and anterior and
posterior border mesoderm from different wing buds. These replicas were
photographed at low magnification to permit determination of the number of
102
R. O. KELLEY AND J. F. FALLON
Table 1
Number of specimens examined
Number of replicas examined
Gap junctions observed/total
number of cells examined*
Number of junctions observed/100
cells
Average number of particles/gap
junction
Tip
Core
Preaxial
Postaxial
U
8
10
9
11
7
12
8
31/256
7/82
16/183
11/151
121
8-5
8-7
7-3
42-4 ±7-6
38-3 ±5-2
37-4 ±6-5
34-5 ±4-7
* Data represent the total number of gap junctions observed/total number of cells
examined in a minimum of seven replicas of each region of limb mesoderm.
cells included in each replica. Thorough analysis of each replica permitted
examination of most, if not all, gap junctions revealed by the fracture plane.
Total numbers of junctions observed per number of cells examined in each
region were recorded and the numbers of particles in gap junctions in each
region were counted directly and averaged.
In this report the terms P-face and E-face particles indicate intramembrane
particles which remain associated with the inner and outer portions, respectively,
of the fractured cell membrane (Branton et ah 1975) Illustrations of replicas
are presented with the platinum shadow direction approximately from the
bottom to the top of the plate.
OBSERVATIONS
Table 1 presents data generated from analyses of electron micrographs of
a minimum of seven replicas from each region of mesoderm examined (subridge, core, anterior and posterior areas). By counting the number of gap
junctions observed in the total number of cells examined in each area, it is clear
that numbers of gap junctions between cells of the mesoderm do not vary widely.
It should be noted that a slight decrease in the number of junctions was apparent
F I G U R E S 2-4
Fig. 2. Surface replica of a freeze-fractured cell in sub-ridge mesoderm. Three gap
junctions (gj) are characterized by aggregated P-face particles (p) and an hexagonal
pattern of pits (arrows) in the attached E-face of the apposed cell, x 100000.
Fig. 3. Profile of a gap junction (arrows) between apposed bodies of mesenchymal
cells, x 25000.
Fig. 4. Replica of gap junction observed between bodies of apposed sub-ridge cells.
Note channels (arrows) which interrupt the particle lattice creating smaller domains
within the perimeter of the junction, x 100000.
Gap junctions in limb-bud mesoderm
104
R. O. KELLEY AND J. F. FALLON
in the posterior border of the limb which may reflect changes preceding death
of cells in the posterior necrotic zone. In addition, the average number of
particles in individual gap junctions varies little when comparisons are made
among regions. From these data, gap junctions appear to have approximately
the same distribution throughout all regions of limb mesoderm during stages
22-24. To avoid redundancy in the text, we present micrographs representative
of gap junctions observed throughout limb bud mesoderm.
Figure 2 illustrates both P and E fracture faces of a typical mesodermal cell.
Three gap junctions are present in the fractured membrane. Each junction is
characterized by aggregates of P-face particles, approximately 8-9 nm in
diameter, and portions of the attached E-faces of the apposed cell membranes.
An hexagonal lattice of pits exists in each E-face with center-to-center spacings
of approximately 8-5-9-5 nm. Although precise configuration of cell-to-cell
association is not certain, it is probable that junctions illustrated in Fig. 2 are
between closely apposed bodies of mesenchymal cells. A gap junction between
the bodies of two mesenchymal cells is clearly illustrated in Fig. 3. Although
the junction is near the nucleus of the cell depicted, no general pattern of
distribution has been observed for the position of assembled junctions. Similar
profiles have been identified between closely apposed cells throughout the
entire limb mesoderm. On occasion, we have observed that the lattice of 8-9 nm
particles comprising a gap junction is transected by narrow channels, forming
small, separate domains of particles within the larger periphery of the junction
(Fig. 4). It is noteworthy that gap junctions with a dispersed particle lattice
appear to be most prevalent in the sub-ridge mesoderm.
Gap junctions were also observed to develop between elongated cellular
processes and similar projections from neighboring cells. Figure 5 shows
a junction connecting the tip of one process with the lateral surface of another.
We have also observed gap junctions between both the apposed tips of cell
processes and the sides of projections which are in contact. Intramembrane
organization characteristic of gap junctions at the tips of cell processes is further
confirmed by the freeze-fracture image illustrated in Fig. 6. In addition, gap
FIGURES
5-8
Fig. 5. Gap junction (arrows) at point of contact between individual cell processes.
The tip of one projection is in contact with the lateral surface of the apposed process,
x 25000.
Fig. 6. Intramembrane organization characteristic of gap junctions at tip of cell
process. Two gap junctions (arrows) are visible, x 57000.
Fig. 7. Tip of cell process in contact with body of adjacent mesodermal cell. Note gap
junction (arrows) at point of contact, x 30000.
Fig. 8. Replica of fractured tip of cell process. The E-face of the cell membrane at the
tip of the process is exposed, revealing an hexagonal pattern of pits characteristic of
gap junctions (arrows), x 85000.
Gap junctions in limb-bud mesoderm
s«ii
105
106
R. O. KELLEY AND J. F. FALLON
junctions form at the tips of processes which traverse the extracellular matrix
and contact the bodies of neighboring mesenchymal cells. Figure 7 shows the
tip of a cell process in contact with the body of an adjacent mesenchymal cell.
Higher magnification reveals that apposed membranes exhibit a gap between
outer leaflets of approximately 3-5-4-5 nm. By fracturing through the tips of
such structures, the E-face of the cell membrane at the tip of a process may be
exposed to reveal a small lattice of pits characteristic of the gap junction
(arrows, Fig. 8).
Upon examination of the sub-ridge region with the scanning electron microscope, we found that processes not only extend short distances between cells
but may also extend several micrometers through the extracellular matrix
(Figs. 9 and 11). Thus there is contact and potential coupling of cells several cell
diameters distant from one another in the sub-ridge mesoderm. This is less
common in other regions of the limb mesoblast where numerous, shorter
cellular projections extend to neighboring, more densely packed cells (Fig. 10).
It should be noted that although processes are often observed inclose association
in. all parts of the limb bud, gap junctions are not always observed at points
of apparent contact.
DISCUSSION
The observations presented in this report show that cells throughout the limb
mesoderm develop gap junctions and are potentially capable of intercellular
communication. Gap junctions were observed at contact points between
adjacent cell bodies, between cell processes, and between cell processes and
cell bodies. Although gap junctions were found at contact points of cell processes throughout the limb, gap junctions appeared to be most numerous in the
sub-ridge area. Moreover, scanning electron microscopy revealed that numerous
filopodia which establish contact with cell bodies or other filopodia over a distance of several cell diameters were present in the sub-ridge, but were considerably less prevalent elsewhere in the limb mesoderm. The latter observation is
essentially in agreement with the report of Searls et al. (1972) using thin sections
FIGURES
9-11
Fig. 9. Scanning electron micrograph of sub-ridge mesoderm (typical of stages 22-24).
Elongate, cellular projections (arrows) traverse the intercellular matrix to contact
other cells several micrometers distant. Similar projections are less common in
deeper mesoderm. x 5500.
Fig. 10. Mesoderm in the core of a limb bud (stages 22-24). Cells are more closely
associated than in the sub-ridge and processes traverse shorter intercellular distances
to apposing cells, x 5500.
Fig. 11. Higher magnification of sub-ridge mesoderm. Note examples of contact
between projections of one cell and a process from an adjacent cell (larger arrow);
and between a cell process and the cell body of an apposed cell (smaller arrow),
x 20000.
Gap junctions in limb-bud mesoderm
107
108
R. O. KELLEY AND J. F. FALLON
of developing chick limb cartilage. It is reasonable to suggest that cell processes
of the limb mesoderm may be correlated with probing and exploratory behavior
of individual cells (Albrecht-Buehler, 1976). Moreover, we have demonstrated
that gap junctions often appear at these discrete points of intercellular contact.
This suggests that gap junctions may couple and uncouple as cells probe and
establish subtle alterations in their individual positions within the developing
limb matrix.
The presence of gap junctions and the electrotonic or metabolic coupling of
cells are coincident (e.g. Johnson, Hammer, Sheridan & Revel, 1974) in all but
a few cases (e.g. Bennett & Trinkaus, 1970). Furthermore, the disruption of gap
junctions (e.g. Barr, Dewey & Berger, 1965) causes the uncoupling of intercellular communication. It appears then that gap junctions mediate cell-to-cell
coupling. Thus there is at least the potential for all, or most of the limb bud
mesoderm cells to be electrotonically or metabolically coupled. However, it
must be recognized that all junctions may not be functional even though they
are structurally developed. Several investigators have suggested that it is
possible for gap junctions to be assembled between contacting membranes but
not be permeable to ions and metabolites (e.g. Albertini, Fawcett & Olds, 1975).
Recent work by Peracchia & Dulhanty (1976) and Peracchia (1977) suggests
the possibility that gap junctions with closely aggregated particles are not
permeable (hence the cells are uncoupled), whereas a slight dispersion of
particles in the hydrophobic plane of the membrane is a structural indication
of permeability (i.e. cellular coupling; cf. Fig. 4).
It is far from clear what the precise roles of gap junctions and concomitant
intercellular communication are in development and morphogenesis of any
developing system. Of the several possibilities, one consideration is that
initiation of differentiation may be related to the assembly and maintenance of
gap junctions which permit constant electrotonic or metabolic signalling between
cells. Alternatively, interruption or reduction of signals either mediated or
controlled by gap junctions may be a principal component in the mechanisms
of differentiation (cf. Loewenstein, 1968; Lopresti, Macagno & Levinthal, 1974;
Griepp & Revel, 1977). We have recently begun an analysis of surface area
occupied by particles of gap junctions between mesodermal cells in the limb
regions. From the preliminary information we have, it appears that a given gap
junction occupies a greater surface area in the membranes of cells in the subridge mesoderm than in plasma membranes in other regions of the limb.
Considering that the number of particles per gap junction is similar in all
regions of limb mesoderm, this apparent reduction of membrane surface area
occupied by gap junctions in more proximal limb regions suggests that the
particle lattice of each structure is more densely organized. In view of the reports
of Peracchia (discussed above), one would predict that a reduction in the degree
of intercellular coupling develops as cells become positioned more distant to
the influence of the apical ridge.
Gap junctions in limb-bud mesoderm
109
In this context, it is reasonable to hypothesize that gap junctions are regulating
as well as communicating structures for intercellular signals in the developing
limb, and that the apical ridge ultimately conditions the strength of the signal.
Tight coupling may be the basis for maintaining the embryonic nature of the
sub-ridge (Searls, 1965) whereas too rapid uncoupling as a result of ridge removal
(Cairns, 1969) may bring about cell death in sub-ridge mesoderm. In
addition, such changes in signals through gap junctions may be an underlying
mechanism in establishing the cell behavior which leads to differentiation as
proposed in the progress zone theory of limb development (Summerbell et ai,
1973). Experiments must be designed to investigate the nature and levels of
intercellular signalling within limb mesoderm and the significance of such
signals in the morphogenesis of the vertebrate limb.
Grateful acknowledgment is made to Drs Allen W. Clark, Miles Epstein, A. J. Ladman,
Bruce H. Lipton and David Slautterback and Mr Steve Thoms for their helpful discussions
and critical reading of the manuscript; to Ms R. Azad and Mrs B. Kay Simandl for expert
technical assistance; and to Ms Anita Kimbrell and Mrs Debra Reierson for preparation of
the typescript. This investigation was supported by NIH grants AG 00191 and HD 07402;
by NSF grants GB 24704 and GB 40506; and by an institutional grant to the University of
Wisconsin from the American Cancer Society. R.O.K. is the recipient of a Research Career
Development Award from the NIH (HD 70407).
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{Received 20 December 1977, revised 25 April 1978)