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/ . Embryol. exp. Morph. Vol. 57, pp. 3-24, 1980
Printed in Great Britain © Company of Biologists Limited 1980
Intercellular bridges in the embryo of the
Atlantic squid, Loligo pealei
I. Cytoplasmic continuity and tissue differentiation
By JOINER CARTWRIGHT, JR 1 AND JOHN M. ARNOLD 2
From the University of Hawaii, Pacific Biomedical Research Center,
Honolulu, Hawaii
SUMMARY
In the post-gastrulation embryo of the Atlantic squid, Loligo pealei, the cells of the developing blastoderm are joined to each other by intercellular bridges which may provide a means of
cytoplasmic communication between the cells. This paper describes an electron microscope
survey of bridges in the developing blastoderm just prior to, and during, the onset of differentiation. The bridges are similar to those described in the gonadal tissue of many animal
species and appear to result from incomplete cytokinesis followed by the disappearance of the
spindle remnant. The bridges persist and chains of cells result which are generally branched
and coiled. In the undifferentiated blastoderm the chains of cells show no apparent orientation to each other. However, in the apical blastoderm undergoing differentiation, chains of
bridged cells appear to coincide closely with the developing mantle and shell gland primordia.
The configuration a chain of cells assumes depends upon the degree of branching (i.e. the
number of cells having three bridges) and the degree of coiling of the chain. Whereas coiling
is probably affected by the crowding of neighboring cells, both branching and coiling appear
to be functions of spindle orientation relative to previous bridges. During mitosis the bridges
appear to become occluded by systems of transverse membranous cisternae, and mitotic
nuclei are thus isolated. However, the bridges apparently re-open during Gl5 and during
periods of protein synthesis the cells within a group share a common cytoplasm. It is suggested that gene products are shared and protein synthesis of the entire bridged group may be
synchronized. As the sharing of control molecules may also be facilitated, these essentially
syncytial groups may respond uniformly to inducers from the yolk syncytium, or other tissues
and differentiation may be synchronized within the group.
INTRODUCTION
In 1955 Burgos &Fawcett confirmed at the ultrastructural level that incomplete
cytokinesis resulted in cytoplasmic bridges being formed between otherwise
separate spermatids in the cat. Since then intercellular bridges have been described in developing ovaries in mammals (Franchi & Mandl, 1962), insects
(Ramamurty, 1964), crustaceans (Anteunis, Fautrez-Firlefyn & Fantrez, 1966),
1
Author's address: Section of Cardiovascular Sciences, Department of Medicine, Baylor
College of Medicine, Texas Medical Center, Houston, Texas 77030, U.S.A.
"Author's address: University of Hawaii, Pacific Biomedical Research Center, 41 Ahui
Street, Honolulu, Hawaii, 96813.
4
J. CARTWRIGHT AND J. M. ARNOLD
annelids (Anderson & Heubner, 1968), amphibians (Ruby, Dyer, Skalko &
Volpe, 19706), rotifers (Bentfield, 1971), arachnids (Brinton, 1971), fish (Satoh,
1974), and cephalopods (Selman & Arnold, 1977). Similar bridges have also
been described between developing sperm cells in birds (Nagano, 1961), insects
(Hoage & Kessel, 1968), millipedes (Reger & Cooper, 1968), fish (Clerot, 1971),
echinoderms (da Cruz-Landim & Beig, 1976), and gastropods (de Jong-Brink,
Boer, Hommes & Kodde, 1977).
The primary function attributed to bridges in gonads is the synchronization
of cellular events. In the higher animals, germ cells arise from a population of
gonial cells that have proliferated by a series of highly synchronized mitotic
divisions (Fawcett, Ito & Slautterback, 1959; Zamboni & Gondos, 1968;
Gondos & Zamboni, 1969; Dym & Fawcett, 1971; Brinton, 1971; Kinderman &
King, 1973; Woodruff & Telfer, 1973). Following proliferation, the germ cells
undergo meiosis, also in a highly synchronized manner (Fawcett et al. 1959;
Skalko, Kerrigan, Ruby & Dyer, 1972; Satoh, 1974; Filosa & Taddei, 1976;
Brinton, 1971). In most male animals, meiosis is followed by synchronized
maturation of the spermatozoa, during which time the cells remain bridged in
clusters. This association between intercellular bridges and synchronization of
maturation has been described in the testis of coelenterates, birds, insects (Fawcet
et al. 1959; Dym & Fawcett, 1971), the opossum, rabbit and man (Fawcett et al.
1959), chinchilla, bull, ram (Dym & Fawcett, 1971), and the cat (Burgos &
Fawcett, 1955; Dym & Fawcett, 1971). Similarly, bridges have been implicated
in the synchronization of maturation in the ovaries of several animals including
the tick (Brinton, 1971), the mouse (Ruby, Dyer & Skalko, 1969) and the rabbit
(Gondos & Zamboni, 1969). During the development of the gonad in the above
animals, the germ cells remain joined in clusters by intercellular bridges, and it
is believed that the resulting cytoplasmic continuity allows for the distribution
of control molecules and is responsible for the synchrony of both division and
maturation. Reminiscent of the bridged cells in the testis are the developing
nematocysts in Hydra (Fawcett et al. 1959). Synchronous division of somatic
interstitial cells results in clusters of 14-16 cnidoblasts, linked by intercellular
bridges, which then undergo synchronous maturation into nematocysts.
Regulation of the numbers of maturing germ cells by systematic cellular
degeneration has been reported in female rabbits (Zamboni & Gondos, 1968;
Gondos & Zamboni, 1969), mice (Ruby et al. 1969) and humans (Ruby, Dyer,
Gasser & Skalko, 1970 a; Gondos, 1973 a), and male monkeys (Gondos &
Zemjanis, 1970; Dym & Fawcett, 1971), rats, guinea-pigs, Chinese hamsters,
chinchillas, cats, cattle, and sheep (Dym & Fawcett, 1971). The mechanism for
this degeneration is unknown, but an association with cell division has been
noted and intercellular bridges have been implicated in synchronizing its onset
(Zamboni & Gondos, 1968; Gondos & Zemjanis, 1970; Dym & Fawcett, 1971;
Gondos, 1973 a). Another function attributed to intercellular bridges is that of
intercellular transport of nutrient materials. Evidence for this function is based
Intercellular bridges in the embryo of the squid
5
primarily upon studies of the nurse cell-oocyte relationship in invertebrates
(Koch & King, 1966 and 1969; Anderson & Heubner, 1968; Cassidy & King,
1969; King & Akai, 1971; Woodruff & Telfer, 1973) in which one cell in a cluster
of bridged oogonia is destined to mature into an ovum while the others transfer
nutrients and organelles to the oocyte through the bridges. Intercellular bridges
are also thought to restrict mitosis in the ovary of insects. In most insects, gonial
cells (cystocytes) undergo division in a precise and predictable pattern, yielding a
cluster containing a species-specific number of cells attached to each other by
bridges. After this pattern is achieved, mitosis stops and the cell with the
requisite number of bridges begins differentiation to an ovum (King & Akai,
1971; Johnson & King, 1972; Kinderman & King, 1973). In his review of intercellular bridges in mammalian germ cell differentiation, Gondos (1973 b)
suggested that bridges serve to restrict the mobility of cells. Although groups of
bridged cells migrate, bridging may retain certain essential spatial relationships.
Nagano (1961) showed that the intercellular bridges between spermatocytes
in the rooster may become temporarily closed by a system of membranous
cisternae forming across the channel. Dym & Fawcett (1971) described similar
occluding membranes in bridges between spermatocytes in the ram, rat, guineapig, cat, chinchilla and Chinese hamster, and bridges between spermatogonia in
the ram and the rat. These authors speculated that the purpose of the occluding
membranes was to isolate certain cells from the germinal syncytium and the
degeneration process. They also noted that when bridges became occluded
synchrony of mitotic events among the cells was less apparent (Dym & Fawcett,
1971).
Recently Arnold (1974) reported that intercellular bridges occurred between
the future somatic cells of the blastoderm in the embryo of the squid, Loligo
pealei. The bridges appeared to be very similar in morphology to those described
in the gonad of most animals, and were occasionally found to be occluded by
transverse membranous cisternae in a manner similar to that described by
Nagano (1961) and Dym & Fawcett (1971). The purpose of this paper is to
describe the distribution of intercellular bridges in the Loligo pealei embryo and
relate the resulting cytoplasmic continuity to certain aspects of the development
of the organism.
MATERIALS AND METHODS
The Loligo pealei embryos used in this study were obtained by inducing adult
animals to spawn (Arnold, 1962) in tanks of running sea water at the Marine
Biological Laboratory at Woods Hole, Massachusetts. The strings of embryos
were kept in their jelly in running sea water at 16-20 °C until they reached midblastoderm stages 16 and 17 (references to embryonic stages in Loligo pealei are
from Arnold, 1965 a). They were mechanically removed from the egg jelly and
dechorionated with jeweler's forceps and fine iridectomy scissors. The embryos
were then fixed by a procedure modified from that of Palade (1952) in which they
J. CARTWRIGHT AND J. M. ARNOLD
Section plane
Margin ot
blastoderm
(a)
Laterally sectioned
embryo, stage 16
(b)
Apically sectioned
embryo, stage 17
Fig. 1. Diagram showing the areas sectioned for the embryo surveys. In the laterally
sectioned embryo (a) the area surveyed was just toward the animal pole from the
advancing margin of the blastoderm. In the apically sectioned embryo (b) the
surveyed area was at the animal pole. Scale bar: 0-5 mm.
were immersed for 15 min in 1-0% osmium tetroxide in 0-25 molar veronal
acetate buffer adjusted to pH 6-8-7-0 at 20-22 °C. This was followed by several
changes of 50 % ethanol, dehydration through a graded series of ethanol into
propylene oxide and embedding in Epon according to Luft (1961).
To determine the distribution of intercellular bridges, and the linkage patterns
of bridged cells in the blastoderm, three-dimensional reconstructions were made
of limited areas of the blastoderm from electron micrographs of serial thin
sections. In the first survey, sections were cut from the undifferentiated blastoderm, just toward the animal pole from the advancing blastoderm margin, of a
stage-16 embryo (Fig. la). In the second survey, sections were cut from the
animal pole of a stage-17 embryo (Fig. \b) where the mantle and shell gland
primordia were in the early stages of differentiation (Arnold, 1971). Each embryo
was trimmed and oriented in the ultramicrotome so that serial sections could be
cut tangentially to the surface of the embryo. Sections were collected and
mounted on formvar support films on slotted grids until both blastoderm layers
and the yolk syncytial layer were sectioned completely through over a wide area.
These were stained with uranyl acetate (Stempak & Ward, 1965) and lead
citrate (Venable & Coggeshall, 1965) and examined with the electron microscope,
and the entire area of each section was photographed. The plates were printed at
a magnification of 2250 x and the prints were montaged to make single images
of each section, which were then carefully examined for intercellular bridges.
Intercellular bridges in the embryo of the squid
7
The location of each bridge relative to the cells was marked on each section even
though the bridge did not appear in all sections. The cell outlines of every tenth
section were then traced on paper (Figs. 2 through 7) and the location of each
bridge was marked on each tracing. The groups of bridged cells were indicated
on the tracings with transfer design patterns. Ninety-six serial sections were
required for the laterally sectioned embryo, and the diagrams of the 42nd (Fig. 2),
52nd (Fig. 3) and 70th (Fig. 4) sections are presented here. One hundred and
seventy-six sections were required from the thicker, differentiating blastoderm
of the apically sectioned embryo. Diagrams of the 60th (Fig. 5), 100th (Fig. 6)
and 176th (Fig. 7) sections are presented here. Figure 8 shows the 100th section.
RESULTS
The cells in both the undifferentiated blastoderm (laterally sectioned embryo)
and the blastoderm undergoing differentiation (apically sectioned embryo)
appeared to be joined to one or more neighbors by intercellular bridges very
similar to those described in gonadal tissue (see Introduction). The cells were
linked in coiled chains that in the undifferentiated blastoderm showed no obvious
orientation to each other, but in the differentiating blastoderm appeared to
coincide with developing organ primordia. Often the chains of cells were
branched, i.e. one or more cells had three bridges. However, in neither embryo
did there appear bridges joining cells of different cell layers.
There were mitotic cells in both embryos that were bridged to cells in differing
stages of mitosis or to interphase cells. In each case the bridges associated with
mitotic cells appeared typical except that, arranged across the channel of each
one was a series of transverse membranous cisternae that appeared to interrupt
the cytoplasmic continuity between the bridged cells. Therefore although mitotic
cells were bridged to other mitotic cells, or to interphase cells, they appeared to
be isolated cytoplasmically from those cells. Several other bridges, not associated
with mitotic cells, were also of the occluded type.
Normal development of Lo/igo pealei has been described (Arnold, 1971;
Arnold & Williams-Arnold, 1976). The surveys presented here are of embryonic
stages just prior to the onset of tissue differentiation (the laterally sectioned
embryo) and just after tissue differentiation has begun (the apically sectioned
embryo). At these stages the embryo consists of three layers covering the apex
of the large ovate mass of yolk like a cap. The innermost is a syncytial yolk
epithelium that is continuous with a thin layer of cytoplasm entirely surrounding
the yolk. Covering the yolk epithelium are the middle and outer cell layers, each
one cell deep. As development progresses, the entire complex extends downward
from the apex until the entire yolk mass is covered with cells. As the blastoderm
completely covers the yolk, organ primordia begin to appear at the apex as
thickenings, or invaginations, of the outer two layers.
The diagrams in Figs. 2 through 7 represent three-dimensional surveys of areas
J. C A R T W R I G H T AND J. M. ARNOLD
FIGURES
2-4
Diagrams of sections of the laterally sectioned embryo showing the groups of bridged
cells. The numbers refer to cell groups specifically referred to in the text. Groups 8
through 16 are in the middle cell layer. The others are in the outer layer. M = metaphase cell; A = anaphase cell. Scale bars: 20 /im.
Fig. 2. Diagram of the 42nd section of the laterally sectioned embryo.
Intercellular bridges in the embryo of the squid
ft M '";;
Fig. 3. Diagram of the 52nd section of the laterally sectioned embryo.
10
J. C A R T W R I G H T AND J. M. ARNOLD
Fig. 4. Diagram of the 70th section of the laterally sectioned embryo.
Intercellular bridges in the embryo of the squid
Figs. 5-7. Diagrams of the apically sectioned embryo showing the groups of bridged
cells. The numbers refer to cell groups specifically referred to in the text. Groups 6
through 17 are in the middle cell layer. The others are in the outer layer. M = metaphase cell; A=anaphase cell; EA = early anaphase cell; LA = late anaphase cell:
T = telophase cell. Scale bars: 20 ju,m.
Fig. 5. Diagram of the 60th section of the apically sectioned embryo. Cell with four
bridges is denoted (*).
11
12
J. CART W R I G H T AND J. M. ARNOLD
Fig. 6. Diagram of the 100th section of the apically sectioned embryo. See also Fig.
Cell with four intercellular bridges is denoted (*).
13
Intercellular bridges in the embryo of the squid
Fig. 7. Diagram of the 176th section of the apically sectioned embryo.
EMB 57
14
J. CARTWRIGHT AND J. M. ARNOLD
of the blastoderm of Loligo pealei embryos compiled from serial sections cut
tangentially to the embryo surface. The groups of cells bridged to each other are
shown as different design patterns. Both outer and middle cellular layers are
included and are distinguished in the diagrams by the design pattern used.
Because of the domed shape of the cell layers, the plane of section cut through
the outer cell layer over an expanding area and then into the middle cell layer.
As sectioning proceeded, the outer cell layer appeared as a ring of cells around
the middle cell layer which appeared as a ring of cells around an area of yolk. In
the case of the undifferentiated blastoderm, however, part of the tissue had
pulled away from the area to be sectioned during specimen preparation, resulting
in a crescent-shaped area of cells with the outer layer forming a semicircle around
the cells of the middle layer (Figs 2, 3 and 4). In the diagrams in which cells
appear separated by a space, or intervening cell, but lie against each other and
are bridged in other sections, the linkage is indicated by a dotted line.
The area of the first survey (laterally sectioned embryo, Figs 2, 3 and 4) was
in the undifferentiated region of a stage-16 embryo just behind the advancing
margin of the blastoderm, about halfway down the embryo from the animal
pole (Fig. 1 a). For the second survey (apically sectioned embryo, Figs 5,6 and 7),
a stage-17 embryo was sectioned tangentially to the animal pole in the vicinity
of the differentiating mantle such that the plane of section was perpendicular
to the long axis of the embryo (Fig. Ib). Figure 8 is a micrograph showing the
100th section from the survey of the apically sectioned embryo. Figure 9 shows,
at higher magnification, a chain of mitotic cells seen in Fig. 8, as well as the
occluded bridges that join them. The first differentiation evident with the light
microscope in the Loligo pealei embryo is the invagination of the shell gland and
early differentiation of the mantle at the apical end in stages 16 and 17 (Arnold,
1971).
In the surveyed area of the laterally sectioned embryo, the outer cell layer
consisted of cuboidal cells of uniform size and morphology averaging 15-7 jum
diameter throughout the area. The cell profiles varied from round and polygonal
to oblong, with few intercellular spaces. The cells of the middle cell layer
appeared similar in morphology to those in the outer cell layer except that the
former were larger, flatter, also undifferentiated and of relatively uniform
diameter of about 21-0 /an. These cells were interspersed with intercellular spaces
of varying size and shape. Cells in both layers had cytoplasmic processes that
extended to other cells and to the yolk epithelium.
In the outer cell layer, there were 104 intercellular bridges linking 130 cells
in 25 groups (Figs 2, 3 and 4). The largest of these groups (group 2) had 20 cells
linked with 19 bridges, and the next largest (group 7) had 15 bridges linking 16
cells. The remaining groups had fewer bridges, and eight groups had only two
cells joined by a single bridge. In the middle cell layer of the area surveyed, there
were 42 bridges connecting 51 cells in nine groups (groups 8 through 16; Figs 2
3 and 4). One group (group 10, Fig. 4) had 15 bridges linking 16 cells. There was
Intercellular bridges in the embryo of the squid
Fig. 8. The 100th serial section in the survey of the apically sectioned embryo shown
diagrammatically in Fig. 6. Area in the rectangle is shown in higher magnification as
Fig. 9. Scale bar: 20/tm.
15
16
J. CARTWRIGHT AND J. M. ARNOLD
one group (number 8) of nine cells linked by eight bridges. The remaining groups
had seven cells or less and three had only two cells. In the outer layer, groups 1
through 7 had one or more branches and in the middle cell layer, groups 9 and
10 were each branched. There were rive mitotic cells in the area surveyed, three
of which were bridged to other cells. In each case the bridges associated with
mitotic cells appeared occluded by transverse cisternae.
In the case of the apically sectioned embryo, the area covered was larger than
that of the laterally sectioned embryo and included 383 cells in both layers. The
cells in this survey appeared to have a very similar morphology to those in the
laterally sectioned embryo except that in the outer layer there seemed to be a
gradual change in the cell diameter from one end of the surveyed area to the
other (Fig. 5). The gradation in cell diameter appears to be unrelated to the
bridging patterns and is probably a result of stretching of the blastoderm, or a
slight misalignment of the knife during sectioning. The surveyed area was
dominated in the outer layer by group number 1, consisting of 112 cells linked by
111 bridges (Figs 5, 6 and 7). The next largest group was number 3, which had
36 cells connected by 35 bridges. The remaining groups of the outer cell layer
had fewer than 19 cells, and nine of these groups had only two cells and one
bridge each. The middle layer in the surveyed area had 63 cells linked by 51
bridges in 12 groups (groups 6 through 17, Figs 5, 6 and 7), including five twocell groups. The largest group in the middle layer was group 6, which had 23
cells (Figs 6 and 7). The rest of the middle layer groups had fewer than ten cells.
There was extensive branching in the groups of both cell layers, and one cell in
group 1 had four intercellular bridges (Figs 5 and 6). As in the laterally sectioned
embryo, there was no apparent bridging between the outer and middle cell layer.
FIGURE 9
The 100th section in the survey of the apically sectioned embryo; higher magnification of the area outlined in Fig. 8. Six cells in different stages of mitosis are joined by
occluded intercellular bridges. A pair of interphase cells is joined by an open bridge.
Scale bar: 100/on.
Fig. 9B. Occluded bridge in the 114th section, located at (b), that joins a late anaphase cell to a telophase cell. 22900 x .
Fig. 9C. Occluded bridge in the 93rd section, located at (c), that joins two late
anaphase cells. 22900 x .
Fig. 9D. Occluded bridge in the 100th section, located at (d), that joins an early
anaphse cell to a late anaphase cell. 22 900 x .
Fig. 9E. Occluded bridge in the 93rd section, located at (e), that joins an early anaphase cell to a metaphase cell. 22900 x .
Fig. 9F. Open bridge in the 100th section, located at (/), joins two interphase cells.
22900 x .
Fig. 9G. Occluded bridge in the 102nd section, located at (g), joins two metaphase
cells. 22 900 x.
Intercellular bridges in the embryo of the squid
17
18
J. CARTWRIGHT AND J. M. ARNOLD
In the surveyed area of the apically sectioned embryo, there appeared 21
mitotic cells, three of which were in the middle cell layer. There were 30 bridges
associated with the mitotic cells and all were occluded by transverse membranes.
Six of the mitotic cells appeared to be bridged only to interphase cells, while each
of the others was bridged to at least one other mitotic cell. There was no apparent
synchronization between mitotic cells bridged to each other. In group 3 there
were six cells linked to each other (Figs 5, 6, 8 and 9) and all were in different
stages of mitosis from metaphase to late telophase. Of a random sample of 71
bridges not associated with mitotic cells, 15 (21 %) were occluded, 17 (24 %) had
mid-bodies and 39 (55 %) were open.
DISCUSSION
The results of these investigations indicate that intercellular bridges that
result from incomplete cytokinesis, similar to those described in gonadal tissues
(see Introduction) provide a possible method of communication between cells of
the squid embryo. Arnold (1965/?, 1968) and Arnold & Williams-Arnold (1976)
have shown that in Loligo pealei there is established a pattern of information in
the egg cortex, and later in the yolk syncytium, that directs differentiation in the
overlying blastoderm. They postulate that this information is possibly in the
form of RNA messages which directly, or indirectly, control the selective activation of genetic material during cellular differentiation (Arnold & WilliamsArnold, 1976). Various types of cellular interactions, including gap junctions
and intercellular bridges, provide possible routes for metabolic communication
between the yolk syncytium and the cells of the blastoderm, and among the cells
themselves. Potter, Furshpan & Lennox (1966) showed that in the embryo of
Loligo pealei, up until a few days before hatching, there is a low-resistance ionic
coupling between cells of various organs and the yolk syncytium. Afterwards
this coupling is lost. Although intercellular bridges could provide low-resistance
channels for such coupling, it is likely that gap junctions are also involved, as
there do not appear to be bridges between cell layers or between either cell layer
and the yolk syncytium.
Intercellular bridges might, however, provide certain advantages in communication between cells of the same layer. The bridges would allow passage
of all molecules that can pass through gap junctions, plus larger molecules, such
as preformed RNA and polysomes. They could also allow passage of organelles
such as elements of the cytoplasmic membrane system and mitochondria (Arnold,
1974). Although the bridges appear to be occluded during mitosis, they are
apparently open in Gx and during protein synthesis and DNA replication. Gene
products may then be shared between cells, and protein synthesis of the entire
bridged group might therefore be synchronized. Passage of control molecules
might also be facilitated and these essentially syncytial groups might respond
Intercellular bridges in the embryo of the squid
i
19
II
Fig. 10. This diagram shows how the orientation of the mitotic spindle relative to
previous bridges may cause branching of a chain of cells. If a cell that has two
previous bridges divides so that the division furrow lies between the previous bridges
(1), each daughter cell gets one and a straight chain results (III). If the orientation of
the spindle is such that the previous bridges lie on the same side of the furrow (II),
one of the daughter cells gets both previous bridges. These, with the bridge from the
present division, will give it a total of three (IV) and a branch in the chain results.
uniformly to inducers from the yolk syncytium, or other tissues. This might
allow differentiation within the group to be highly synchronized.
The cells in the differentiating blastoderm appear to be bridged in groups
which closely coincide with developing organ primordia. The apically sectioned
embryo was oriented so that the differentiating mantle and shell gland would
occupy the center of the sectioned area (Arnold, 1965a, 1971). Cells of group 1
surround, in an interrupted ring, the cells of group 2 (Fig. 5). This configuration
strongly suggests that group 2 (and only group 2), gives rise to the shell gland and
that group 1 similarly gives rise to the mantle primordium (see also diagrams of
Naef, 1928, Kollicker, 1844, etc.) because of the exact coincidence of the organ
primordia and these groups of bridged cells. It seems likely that these groups will
develop differently from each other because of their different positions relative
to the yolk syncytium (Arnold, 19656,1968; Arnold & Williams-Arnold, 1976).
However, because of the intercellular bridges, the cells within each group would
differentiate synchronously into their respective organs. Because of the extremely
laborious task of completely serially sectioning and analyzing the tissues, other
organ primordia were not subjected to complete analysis, but enough bridged
20
J. CARTWRIGHT AND J. M. ARNOLD
cells were found in the developing retina and the otocyst primordium to suggest
a similar situation exists in these tissues also.
The configuration a group of cells assumes depends upon the degree of branching of the chain of cells (i.e. the number of cells having more than two bridges)
and the degree of coiling of the chain. Whereas coiling is probably affected by
the crowding of neighboring cells, both branching and coiling appear to be
functions of spindle orientation relative to previous bridges. If the spindle of a
dividing cell is oriented so that the plane of division lies between two previous
bridges (Fig. 10-1), each daughter cell will get one of the previous bridges and
the chain will remain unbranched (Fig. 10-111). Group 4 of the apically sectioned
embryo is such an unbranched chain (Figs 5 through 7). If, however, the spindle
is oriented so that both previous bridges lie on the same side of the division plane
(Fig. 10-11), one of the daughter cells will receive both previous bridges. These
two bridges, plus the one forming from the present division, will result in that
particular cell having three bridges and the chain will be branched (Fig. 10-IV)
such as groups 1 and 2 of the apically sectioned embryo (Figs 5 and 6). If any
division within a chain occurs such that the plane of division is perpendicular to
a line connecting two previous bridges, the division will result in the extension of
a straight chain. Such appears to have been the case in group 4 of the apically
sectioned embryo (Figs. 5 through 7) and most of the divisions in group 10 of
the laterally sectioned embryo (Fig. 4). However, it appears that the end cell of a
chain can divide with the spindle rotated so that the furrow occurs near the
previous bridge. The close proximity of the two bridges would result in a bend
or coiling of the chain. Such appears to have bsen the case in group 5 of the
apically sectioned embryo (Figs 5 through 7), and group 10 of the laterally
sectioned embryo (Fig. 4). There did appear one cell with four bridges in group 1
of the apically sectioned embryo (Figs 5 and 6). This probably resulted when a
cell with three previous bridges divided in such a way that all three bridges were
on the same side of the division plane.
The relationship between the mitotic apparatus and the plane of cleavage has
been formalized as 'Balfour's Rules' (cited in Wilson, 1925; Arnold, 1976) which
state: (1) the plane of cleavage is perpendicular to that of the previous division,
and (2) the plane of cleavage is perpendicular to the long axis of the spindle. The
many cases in which bridges appear on roughly opposite sides of the cell (group
4, Fig. 6 and group 10, Fig. 4) suggest that the first rule is violated in the squid
blastoderm. The second rule, however, provides the basis for the control of
branching and coiling, and therefore the shape of the cell group. Despite the
extensive branching and coiling that characterize the groups of cells, in neither
of the surveyed embryos did there appear a cell in the outer cell layer bridged to
one in the middle cell layer. That the intercellular bridge arises as a result of
mitosis is supported by the observation that in neither embryo did there appear
a chain of cells bridged back upon itself in a circle, and that in every cell group
the number of cells exceeded the number of bridges by one.
Intercellular bridges in the embryo of the squid
21
Even though cells joined by intercellular bridges share a common cytoplasm,
regional differences in morphogenic, or control substances, may occur and
responses may vary within the bridged group. Moens & Hugenholtz (1975)
presented evidence that in the rat, individual spermatogonia in a syncytial group
may initiate differentiation at different times. In the insect ovary, one cell of a
bridged group matures into an ovum while the others become nurse cells
(Woodruff & Telfer, 1973). In the squid blastoderm mitosis is not synchronized.
It appears that when an embryonic somatic squid cell enters mitosis, the bridges
that link it to neighboring cells become occluded, thereby interrupting cytoplasmic continuity and allowing mitotic asynchrony. Throughout both embryos
surveyed, cells in different phases of mitosis are bridged to each other as well as
to interphase cells. An example of this can be seen in group 3 of the apically
sectioned embryo (Figs 5, 6, 8 and 9) in which six cells, all in different phases
of division, are evident. However, at high magnification, all of the bridges
associated with the mitotic cells are seen to be of the occluded type, and there
was no evidence of cytoplasmic continuity between a mitotic cell and any other
cell. Dym & Fawcett (1971) made similar observations in the testis of the ram.
It seems reasonable to assume that there is some advantage in asynchrony of cell
division in the blastoderm and organ primordia. If divisions were synchronous,
the number of cells in a group would be restricted to twice the number of cells
in the previous cycle. Asynchrony would allow the cell number to be regulated
to satisfy the requirements of group size and shape more easily.
The results of the embryo surveys support the hypothesis that the transverse
membranes of the occluded bridge are temporary structures and that bridges
can open and close repeatedly (Nagano, 1961; Dym & Fawcett, 1971; in contrast
to the view of Arnold, 1974). If bridges are formed by incomplete cytokinesis
during division, and every bridge associated with a cell going into mitosis
becomes occluded, then three or more cells linked in a chain by open bridges
indicates that the occluded bridges re-open after division. There are many such
groups in both embryos surveyed (Figs 2 through 7). In group 1 of the apically
sectioned embryo (Fig. 6), there are mitotic cells separated by 15 interphase cells.
This means at least four cell cycles have occurred since their common parent cell
first divided. In group 3 of the same embryo, there are mitotic cells separated by
six interphase cells, which means they are separated by three cell cycles (Fig. 6).
Since about 25 % of the total number of intercellular bridges are closed at one
time, the bridges remain closed for about one-quarter of the cell cycle. Therefore,,
the bridges associated with these mitotic cells must have closed and opened
previously during the growth of the chains.
The authors wish to thank sincerely Dr Ian Gibbons for the use of his electron microscope and Ms Frances Okimoto, Mrs Sandra Haley and Ms Concepcion Mata for their
expert assistance in the preparation of the manuscript. We also wish to thank Dr Margaret
Ann Goldstein and Mr David Murphy for their helpful discussions and critical review of
the manuscript.
22
J. CARTWRIGHT AND J. M. ARNOLD
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