/ . Embryo!, exp. Morph. Vol. 33, 2, pp. 403-417, 1975
Printed in Great Britain
403
The distribution of non-synaptic
intercellular junctions during neurone differentiation
in the developing spinal cord of the
clawed toad
BY BRIAN P. HAYES 1 AND ALAN ROBERTS 2
From the Department of Zoology, University of Bristol
SUMMARY
The distribution of intercellular junctions, other than synapses and their precursors, has
been described in the developing spinal cord of Xenopus laevis between the neurula and free
swimming tadpole stages. At the neurocoel, ventricular cells are joined in the apical contact
zone by a sequence of junctions which usually has one or more intermediate junctions but
often also includes close appositions, gap junctions and desmosomes. This apical complex is
more diverse than that reported in other vertebrate embryos and between ependymal cells in
the adult central nervous system. Gap junctions are also found between ventricular cells and
their processes near the external cord surface. However, no other special junctions occur in
this location under the basement lamella which surrounds the cord. Punctate intermediate
junctions are generally distributed between undifferentiated and differentiating cells and their
processes but were not found in neuropil after stage 28. These results are discussed in relation
to cell movements during neural differentiation, possible effects on the freedom of movement
of ions and molecules through extracellular pathways in the embryo, and possible intercytoplasmic pathways via gap junctions which may be responsible for the physiologically
observed electrical coupling between neural tube cells.
INTRODUCTION
Unlike cells in many tissues (see, for example, Loewenstein, 1969) neurones
are usually not in electrical contact with their neighbours. They are wrapped
by glial processes and bathed by an extracellular medium which is carefully
controlled, often separately from the extracellular space in the rest of the body.
In contrast to this, the embryonic ectodermal cells which give rise to the
nervous system are coupled to one another electrically and lie in an extracellular medium common to the whole embryo (Sheridan, 1968; Slack &
Warner, 1973). Consequently, some important changes must occur in the
contact relationships between cells in the nervous system during development.
In the first place there must be changes in the pathways from the inside of one
1
Author's address: Institute of Ophthalmology, University of London, Judd Street, London
WC1 9QS.
2
Author's address: Department of Zoology, University of Bristol, Bristol BS8 1UG.
26
E M B 33
404
B. P. HAYES AND A. ROBERTS
cell to the insides of its neighbours (the intercytoplasmic pathways). In the
second place there may be changes in the freedom of movement in the extracellular pathways between the cells.
We have investigated these changes by looking at the junctions that are made
by cells in the spinal cord of Xenopus embryos during the first differentiation
of functional neurones. Neural differentiation starts as the neural tube is
forming at stage 19 of Nieuwkoop & Faber (1956). By the time the animal is
hatched and can swim (stage 35) we can be sure that some neurones are well
differentiated. We have therefore examined this period of development paying
particular attention to any differences which exist between the junctions made
by undifferentiated ventricular cells and cells and processes showing signs of
differentiation. [.Functional nomenclature is reviewed at length by McNutt &
Weinstein (1973). We have used the following terms: tight junction = occludens
junction, gap junction = small subunit gap junction or nexus, intermediate
junction = zonula and fascia adherens and desmosome = macula adherens.
Where membranes are close but it is not clear if the junction is tight or gap,
we have used 'close apposition'.] The distribution of close membrane junctions
has been most carefully examined. This is because zonula tight junctions
(zonula occludentes) are thought to limit extracellular pathways (Bennett &
Trinkaus, 1970; McNutt & Weinstein, 1973) and gap junctions have been
closely correlated with intercytoplasmic pathways for ion movement (e.g.
Bennett, Spira & Pappas, 1972; Joseph, Slack & Gould, 1973; McNutt &
Weinstein, 1973; Payton, Bennett & Pappas, 1969; Sheridan, 1971). Also, it
has been suggested that in Xenopus retina gap junctions disappear when neural
elements differentiate (Dixon & Cronly-Dillon, 1972).
Our results show that differentiated neurones and the ventricular cells that
give rise to them share the ubiquitous punctate intermediate or adherens
junction. However, neurones may lack the gap junctions found between
ventricular cells. The distribution of junctions is discussed in relation to pathways for ion movement, cell movement and differentiation.
MATERIALS AND METHODS
Eggs were obtained from breeding pairs of adult Xenopus laevis by priming
and injecting with chorionic gonadotrophin; they were allowed to develop in
aerated tap water at temperatures between 18 and 22 °C. Embryos were staged
using the normal tables of Nieuwkoop & Faber (1956) and animals at stages
15, 16, 17, 19, 21, 22,23, 25, 27, 28,31,33,35 and 48 were prepared for electron
microscopy as follows.
Embryos were transferred to 10 % Holtfreter's solution where, if necessary,
egg membranes were removed using fine forceps. The embryos were then
immediately immersed in buffered fixative. Large areas of yolky endoderm
were removed from the embryos to aid fixative penetration.
Non-synaptic junctions in developing spinal cord
405
Fixative and post-fixation solutions were made up in 0-05 M sodium cacodylate buffer at pH 7-3 containing 1-1-5% glucose and 0-05% CaCl 2 .6H 2 O.
Embryos were fixed in a solution of 4 % glutaraldehyde and 1 % formaldehyde
prepared from paraformaldehyde for 2 h at room temperature, washed in
buffer and postfixed in buffered 2 % osmium tetroxide for 2-4 h. Several stage-27
embryos were stained 'in the block' with a 2 % solution of uranyl acetate
buffered with sodium hydrogen maleate (Brightman & Reese, 1969). Lanthanum
hydroxide was introduced into the extracellular space of a single stage-27
embryo using fixative, buffer and osmium tetroxide containing 1 % lanthanum
nitrate at pH 7-8. All embryos were dehydrated in ethanol, cleared in propylene
oxide and embedded in Araldite.
The spinal cord was sectioned transversely using a Cambridge ultramicrotome to give ribbons of silver-silver grey thin sections. Cross-sections were
taken: of whole spinal cord at stages 15, 16, 17, 19, 22, 23, 25 and 28, in the
cervical and mid-trunk regions; throughout the cord at stage 27; and in the
cervical tract neuropil at stages 31, 33, 35 and 48. Sections were picked up on
celloidion/carbon-coated copper grids, stained with solutions of 10 % uranyl
acetate in 50% ethanol and alkaline lead citrate (Reynolds, 1963). Sections
stained 'in the block' with uranyl acetate or lanthanum hydroxide were stained
with alkaline lead citrate alone. Sections were examined in an AEI EM6B
electron microscope. Micrographs of cell junctions were usually made at
30000 magnification. The position of each junction and characteristics of the
cellular elements on either side were noted. In some cases junctions were
examined through series of sections making descriptions of their shape possible.
Measurements of junctional length (to 0-1 /mi) and cleft width (to 1 nm)
were made from photographs at 105000 magnification.
RESULTS
Around the neurocoel of the embryonic spinal cord lie the apices of the
ventricular cells of the neural epithelium. These ventricular cells are the ultimate
progenitors of the neurones and macroglial cells in the CNS (see Boulder
Committee, 1970, for nomenclature) and form a complex of junctions like that
in other epithelia. The most obvious and usual feature of this apical contact
region in Xenopus was a variable length of intermediate or 'adherens' junction
(0-03-1-0 /<m long in transverse sections of the cord). In such junctions the cleft
was moderately dense, and 5-20 nm wide, membranes were dense, and this
density graded off into the cytoplasm on each side of the junction (Figs. 1, 2).
Sometimes a single clearly defined junction was present but in other cases a
series of rather ill-defined junctions was found (cf. Figs. 1, 2). Occasionally,
desmosomes with marked cytoplasmic density and associated fibrils occurred
with the intermediate junctions and rarely, as in Fig. 3, a desmosome was found
in place of any intermediate junction. Between the intermediate junction and
26-2
406
B. P. HAYES AND A. ROBERTS
Non-synaptic junctions in developing spinal cord
407
the neurocoel there was usually a short region of close membrane apposition
with some membrane thickening and cytoplasmic density (see Figs. 1-3). A few
similar close appositions occurred in the region of intermediate junctions in
some cases. On the neurocoel side of the intermediate junctions the extracellular
space was often expanded. Our survey has revealed no clear changes in the
junctions of the apical contact region at the neurocoel during the period of
development we have examined. The detailed arrangement of junctions was
very variable, tending to be most diverse when the ventricular cell profiles were
narrowest and contained longitudinally orientated microtubules (Fig. 1).
Gap junctions were found between ventricular cells near the apical contact
zone at the neurocoel and also between cells near the outer periphery of the
spinal cord (see Table 1). They consisted of close appositions of parallel dense
membranes with a narrow cleft. The total width of these junctions (i.e. the
distance from cytoplasm of one cell to that of the other across the junction) was
usually 14-16 nm, and the length from 0-1 to 0-9 /.im in section-stained material.
There were two types of cleft, either a moderately dense structureless region
7-9 nm wide (see Figs. 4, 6) or two longitudinal dense lines separated by 2-3 nm
giving the junction a seven-layered appearance (see Figs. 2, 5). Junctions stained
'in the block' with uranyl acetate were of similar total width (see Table 1).
They showed a well-defined seven-layered substructure with an electron-lucent
'gap' 2-3 nm wide (Figs. 7, 8). In oblique sections a polygonal network with
an approximately 12nm centre to centre spacing of subunits was apparent
(Fig. 9). When lanthanum hydroxide was introduced into the intercellular cleft,
the 'gap' of the junction appeared as a black line about 4 nm wide (Fig. 10).
The 'gap' was crossed by more electron-lucent bars spaced about 10 nm apart.
The total width of the junction was 16 nm, which is comparable with that found
using the other staining methods. The examination of serial sections of these
F I G U R E S 1-3
Ventricular cell junctions near the neurocoel at stage 27.
Scale line 0-1 /*m. a = apposition, d = desmosome, g = gap junction, / = intermediate junction, / = lipid yolk granule, mt = microtubules, nc = neurocoel.
Fig. 1. Two junction complexes between lateral ventricular cells in the cervical spinal
cord. The junction sequence from the neurocoel is, on the left, an apposition, a
desmosome, an intermediate junction and then further down the cell a second
intermediate junction and, on the right, an apposition, a desmosome and a gap
junction. The narrow apical profile in the centre contains longitudinal microtubules. 35000.
Fig. 2. Junctions between dorsolateral ventricular cells in the cervical spinal cord.
The sequence from the neurocoel (top left) is two appositions, an intermediate
junction and a gap junction 0-25 /*m long and with a total width of about 15 nm.
The seven-layered substructure of this junction can be seen in places. 210000.
Fig. 3. Junctions between ventricular cells in the trunk spinal cord. The sequence
from the neurocoel is an apposition and a small desmosome. 105000.
408
B. P. HAYES AND A. ROBERTS
'4
F I G U R E S 4-6
Gap junctions between ventricular cells near the outer margin of the stage-27 spinal cord.
Scale line 01 /tm. bl = basement lamella, g - gap junction.
Fig. 4. Gap junction, 0-3 /tm long and with a total width of 15 nm between lateral
ventricular cells. No other junctions are apparent. The loose basement lamella
surrounding the cord can be seen (bottom left). 105000.
Fig. 5. Gap junction in the tail spinal cord. Only part of the junction (total length
0-75 fim) is shown to illustrate the seven-layered substructure where the total
width is 15 nm. 210000.
Fig. 6. Gap junction between cells, one of which is wrapping a neuronal process
(arrowed) containing microtubules. The basement lamella can be seen outside the
cord and also the absence of special junctions between cells at the periphery.
35000.
Outer margin
Near neurocoel
Near neurocoel
Near neurocoel
Outer margin
Near neurocoel
27
27
27
27
27
27
27
27 i
27]
27
27
27
27
27
27
7
6
6
6
8
8
8
8
9
10
27 J
27
27 \
k
/
|'
At neurocoel \
At cord
periphery
—
—
]
-
15
15
15
16
22
22
1
1
2
3
4
4
5
5
5
5
5
5
6
6
]
Region
Stage
no.
Embryo
Mid-trunk
Anterior
/\nienoi
trunk
Tail
Tail
Tail
Tail
Mid-trunk
Cervical
Cervical
Tail
Tail
Tail
Tail
Tail
Tail
Tail
Anterior
trunk
Cervical
Anterior
trunk
Level
I
r
I
(
16
18
18
17
17
—
16
16
16
18
15
14
15
17
15
15
16
15
15
15
16
14
15
Total
width
(nm)
—
—
—
7 layered
7 layered
—
7 layered
—
7 layered
—
—
—
—
7 layered
—
Substructure
gap
7 layered
polygonal lattice
7 layered 2 nm gap
7 layered 2 nm gap
7 layered 2 nm gap
7 layered 2-5 nm
—
Lanthanum-stained
0-8
4nm gap
0-35
0-6
0-45
012
0-3
0-8
0-6
'In block' stained
0-35
7 layered
0-25
0-4
0-5
0-3
0-75
0-5
0-75
0-9
0-5
0-38
01
0-5
0-25
0-35
0-2
Section-stained
Length
(/*m)
Table 1. Catalogue of gap junctions
Lateral ventricular cells
Ventricular cells
Ventricular cells
Ventricular cells
Dorsal ventricular cells
Dorsal ventricular cells
Dorsolateral ventricular cells
Dorsal ventricular cells
Dorsal undifferentiated processes
Lateral ventricular cells
Deep median neural plate cells
Deep median neural plate cell and a process
Deep median neural plate cells
Deep median neural plate cells
Ventral cells
Ventral processes
Lateral ventricular cells
Dorsal ventricular cells
Lateral ventricular cells
Lateral ventricular cells
Dorsal ventricular cells
Dorsolateral ventricular cells
Dorsolateral undifferentiated processes
Lateral undifferentiated processes
Found between
1
si
^-
T;
Co
o'
Cl
:s
~*
g
^.
s:
410
B. P. HAYES AND A. ROBERTS
Non-synaptic junctions in developing spinal cord
411
junctions showed that they were punctate (i.e. discreet patches in face view)
rather than zones around the cells. Fig. 1 shows the usual position of gap
junctions found between ventricular cells near the apical contact zone. Elsewhere
they were found between cell bodies and processes, and between processes.
Only 24 were found altogether. Three of these were between ventricular cells
near the neurocoel. The rest were between cellular elements which contained
closely spaced cytoplasmic dense granules (probably ribosomes), a few scattered
rough endoplasmic reticulum cisternae, and poorly developed Golgi bodies.
Such contents are typical of ventricular cells and in a number of cases the
elements making gap junctions could be identified as such (Figs. 4, 6), either by
tracing them to the neurocoel or by their contents. Near the periphery of the
spinal cord the ventricular cells contained many dense granules, mitochondria,
smooth-surface tubules and vesicles, and patches of microfilaments and granular
material, like the external processes of ventricular cells in mouse cerebral
vesicle (Hinds & Ruffett, 1971). During neurulation (stages 15 and 16) gap
junctions were found between deep neural plate cells (see Schroeder, 1970) and
their processes. When the neural tube had closed (stage 22) they occurred
between ventrolateral ventricular cells and processes in the cervical spinal cord
(see fig. 8 in Hayes & Roberts, 1973). At stage 27 they were most numerous in
the tail region, although one was found in the anterior trunk and one at the
cervical level of the cord (see Figs. 4, 6). None were found in the tract and
neuropil areas around the margin of the cervical spinal cord after stage 27
although they may have persisted in other parts of the spinal cord which were
not examined. Gap junctions were longest in the tail region at stage 27.
Intermediate or 'adherens'junctions (adherentia of Farquhar & Palade, 1963),
recognized by a moderately dense cleft and dense membranes blending into
amorphous dense cytoplasm on both sides of the junction, were not confined to
F I G U R E S 7-10
Gap junctions in the stage-27 spinal cord.
Figs. 7-8. Stained 'in the block' with uranyl acetate.
Fig. 7. Gap junction in the tail between dorsal ventricular cells near neurocoel. The
total width is 17 nm, the 'gap' is 2 nm wide and the junction is 06 /tm long.
240000.
Fig. 8. Gap junction in the mid-trunk between dorsal undifferentiated processes
near the outer margin of the cord. The total width is 17 nm, the 'gap' is 2-5 nm and
the length 0-35 nm. 240000.
Fig. 9. Gap junction sectioned obliquely showing network of polygonal subunits
with 10-12 nm spacing. This junction is between ventricular cells near outer cord
margin and is 08/tm long. 150000.
Fig. 10. Gap junction, infiltrated with lanthanum hydroxide, between ventricular
cells near the neurocoel in the mid-trunk. Lanthanum hydroxide fills the 'gap' and is
interrupted periodically by more electron-lucent bands crossing the junction. The
'gap' is 4 nm wide and the total width is 16 nm. 240000.
412
B. P. HAYES AND A. ROBERTS
II
F I G U R E S 11, 12. Intermediate junctions in the stage-27 spinal cord.
Fig. 11. Intermediate junction between a ventricular cell and an undifferentiated
process in the tract neuropil area of the trunk. The junction is 0-2 /im long and the
intercellular cleft is 10-14 nm wide. 105000. Scale line, 01 /*m.
Fig. 12. Intermediate junction between ventricular cells in the tail. There is a large
coated vesicle on the right of the junction. The cleft is about 18 nm and the length
0-15 /tm. Scale line, 0-1 jum.
the apical contact zone at the neurocoel. They were also found between cell
bodies, processes and cells, and between processes in all parts of the spinal
cord. Serial sections showed that these generally distributed junctions were
punctate rather than zones. They were found in all regions of the cord examined
at stages up to and including stage 28. However, they were not present in tract
and neuropil areas of stages 31, 33, 35 and 48 cervical cord. The cellular elements
on either side were sometimes undifferentiated ventricular cells (Figs. 10, 11)
or showed signs of neural differentiation such as microtubules, microfilaments
and vesicles of about 50 nm diameter. Junctional lengths were between 0-05
and 0-9 /*m and the mean lengths for all stages were between 0-1 and 0-3 ju,m.
There was no significant increase in the mean junctional length with age; at
stage 15 the mean length was 0-24 + 0-07 /im, at stage 27, 0-19 ± 0-02 fim.
t tests between the two samples showed no significant difference at the 1 %
level with 18 degrees of freedom (t = 0-133). The width of the cleft at these
Non-synaptic junctions in developing spinal cord
Neurocoel
, .w
x,
,
413
,
Plasma
membrane
Close appositions
(zonula)
Intermediate
junction
(zonula)
Gap junction
(puncta)
Extracellular space
Fig. 13. Diagram to show a junctional sequence between ventricular cell membranes
at the neurocoel. The inset relates the diagram to the micrographs of previous figures.
intermediate junctions was between 5 and 25 nm and varied by as much as 15 nm
within an individual junction. No change was detected in cleft width with age.
At the outside margin of the spinal cord there was no evidence of any special
cell junctions under the basement lamella which surrounds the cord (see, for
example, Figs. 4 and 6). The basement lamella was first detected at stage 17
as sparse clumps of fibrillar material. By stage 20, as the neural tube was
closing, it formed a continuous layer around the cord (20-100 nm thick) and
by stage 23 distinct layers of fibrils were present, in places up to 0-5 jam thick.
No further changes were noted up to stage 35. Along the stage-27 cord, steps
in the formation of the basement lamella could be seen, from clumps of fibrils
around the tail spinal cord, to a continuous layer of many striated collagen
fibrils in the anterior trunk region.
DISCUSSION
In the apical contact zone between ventricular cells lining the neurocoel we
have found a series of junctions similar to that in other epithelia. A typical
sequence is shown in Fig. 13 but considerable variations exist in the details of
414
B. P. HAYES AND A. ROBERTS
this sequence. No significant changes were noticed with age and in general the
junctions were very similar to those found between cells in Xenopus embryo
skin (Roberts & Stirling, 1971; Roberts & Hayes, unpublished observations),
Fundulus blastoderm (Lentz & Trinkaus, 1971), amphibian adult skin (Farquhar
& Palade, 1964; Kelly, 1966) and mouse cerebral vesicle, (Hinds & Ruffet,
1971). A somewhat simpler arrangement, lacking close membrane junctions, is
described in the neural tubes of Xenopus (Schroeder, 1970), chick (Fujita &
Fujita, 1963) and mouse (Herman & Kauffmann, 1966). However, in Rana
pipiens Decker & Friend (1974) have described a series of junctions between
ventricular cells at the neurocoel which is similar to that in Xenopus. A point
which still requires clarification in Xenopus is the nature of the close membrane
apposition next to the neurocoel.
Gap junctions in our section-stained material have two distinct appearances;
sometimes the cleft is a moderately dense structureless region 7-9 nm across as
in the junctions of embryonic Xenopus retina (Dixon, 1971; Dixon & CronlyDillon, 1972), sometimes the cleft contains two dark lines with a 2-3 nm separation. In both cases the total width of the junctional membrane complex was
usually 14-16 nm. A 2-3 nm 'gap' has been reported in gap junctions elsewhere
(e.g. vertebrate brain (Brightman & Reese, 1969; Sotelo & Llinas, 1972), mouse
liver (Goodenough & Revel, 1970) and embryonic chick heart (Spira, 1971)).
We therefore checked the membrane spacing in Xenopus cord gap junctions
using 'in the block' uranyl acetate staining. The total width was the same and
the 'gap' was 2-3 nm. In a single junction infiltrated with lanthanum, the 4 nm
'gap' seen was probably a result of staining the outer membrane leaflets. The
gap junctions seen between ventricular cells in Xenopus embryonic spinal cord
therefore fit the criteria for gap junctions established by Brightman & Reese
(1969). They also have lighter bars across the lanthanum-filled 'gap' which
these authors report as typical of interneuronal junctions in adult vertebrates.
We have found gap junctions between ventricular cells in and near the apical
contact zone at the neurocoel, and between ventricular cell processes near the
outer margin of the spinal cord. As the total number of gap junctions was small,
we cannot be sure that they do not exist between other classes of cells. However,
the distribution we have found provides corroboration of previous observations
of Xenopus retina where gap junctions were similarly only found between nondifferentiated cells (Dixon, 1971; Dixon & Cronly-Dillon, 1972). In the spinal
cord and retina gap junctions have not been found so far on differentiating
neurones. This could be because they are even less frequent or that they are
absent altogether.
Small intermediate or adherens junctions have been found generally between
all types of cell and process, both undifferentiated and differentiated. They are
similar to small punctate junctions found in mature nervous tissue (Peters,
Palay & Webster, 1970) and in embryonic Xenopus retina (Dixon & CronlyDillon, 1972), embryonic chick retina (Sheffield & Fischman, 1970; Sheffield,
Non-synaptic junctions in developing spinal cord
415
1971) and spinal ganglia (Pannese, 1968), rabbit dorsal root (Tennyson, 1970)
and mouse cerebral vesicle (Hinds & Ruffet, 1971). Similar junctions in other
animals are said to be temporary (see Pannese, 1968; Tennyson, 1970) and we
have not found them in the tract and neuropil areas of Xenopus after stage 28.
They are the only type of junction that we have found between differentiating
neurones and their processes until synaptic contacts develop.
What then is the significance of the junction distribution in the early Xenopus
spinal cord for movements of ions and molecules in the extracellular and intercytoplasmic pathways? We have found no signs of any junction which would
limit movements across the outer margin of the spinal cord. Extracellular space
within the spinal cord is therefore probably continuous with that in the rest of
the animal at least up to stage 35, the latest we examined. If the close membrane
apposition between ventricular cells at the neurocoel formed a zonula tight
junction all round the necks of the cells, then a barrier could exist here in the
extracellular pathway for movement to and from the neurocoel. Such a barrier
probably exists in Xenopus skin (Roberts & Stirling, 1971). However, in both
places further evidence would be needed to establish this conclusively.
The distribution of gap junctions suggests that intercytoplasmic pathways
are open between ventricular cells, both near the neurocoel and also near the
outer margin of the cord. Other undifferentiated processes may also be coupled
to one another. Very similar gap junctions have been found occasionally between
skin cells in Xenopus where it has also been shown physiologically that the cells
are electrically coupled (Roberts & Stirling, 1971; Roberts & Hayes, unpublished). In the neural tube of Xenopus and the axolotl it has been shown similarly
that unidentified cells are electrically coupled (Warner, 1970, 1973). Our fine
structural evidence on the distribution of gap junctions in the neural tube and
early spinal cord suggests that these coupled cells would be undifferentiated
ventricular cells. Furthermore, the ventricular cells remain coupled by gap
junctions until at least stage 27. As the ventricular cells can extend from the
neurocoel to the outer margin of the spinal cord, physiological probing into
any part of the cord may reveal electrical coupling between them or their
processes.
We have not found gap junctions on cells and processes showing signs of
differentiation into neurones. However, we do not regard this as evidence that
such junctions are absent. Small macula gap junctions are difficult to detect by
conventional transmission electron microscopy and this method can give little
idea of their numbers. The same reservations apply to the suggestion by Dixon
& Cronly-Dillon (1972) that gap junctions disappear from Xenopus central
retina during neural differentiation. It seems more likely that as a general rule,
classes of similar neurones may be interconnected by gap junctions and coupled
to one another electrically, like the horizontal cells in dogfish retina (Kaneko,
1971; Witkovsky & Stell, 1973) or the motorneurones of frogs (Grinnell, 1966).
Such open intercytoplasmic pathways between groups of similar neurones
416
B. P. HAYES AND A. ROBERTS
would have interesting implications for the differentiation of neuronal cell
types (see also Bennett et ah 1972; Furshpan & Potter, 1968).
This work was supported by a grant from the Medical Research Council. We are grateful
to Dr E. M. Deuchar and Prof. E. G. Gray for their comments on drafts of this paper.
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{Received 21 June 1974, revised 9 September 1974)