Development 121, 2219-2232 (1995)
Printed in Great Britain © The Company of Biologists Limited 1995
2219
Duels without obvious sense: counteracting inductions involved in body wall
muscle development in the Caenorhabditis elegans embryo
Ralf Schnabel
Max-Planck-Institut für Biochemie, Am Klopferspitz 18A, 82152 Martinsried, Germany
SUMMARY
During the first four cleavage rounds of the Caenorhabditis elegans embryo, five somatic founder cells AB, MS, E,
C and D are born, which later form the tissues of the
embryo. The classical criterion for a cell-autonomous specification of a tissue is the capability of primordial cells to
produce this tissue in isolation from the remainder of the
embryo. By this criterion, the somatic founder cells MS, C
and D develop cell-autonomously. Laser ablation experiments, however, reveal that within the embryonic context
these blastomeres form a network of duelling cellular interactions.
During normal development, the blastomere D inhibits
muscle specification in the MS and the C lineage inhibits
muscle specification in the D lineage. These inhibitory
interactions are counteracted by two activating inductions.
As described before the inhibition of body wall muscle in
MS is counteracted by an activating signal from the ABa
lineage. Body wall muscle in the D lineage is induced by
MS descendants, which suppress an inhibitory activity of
the C lineage. The interaction between the D and the MS
lineage occurs through the C lineage. An interesting feature
of these cell-cell interactions is that they do not serve to discriminate between equivalent cells but are permissive or
nonpermissive inductions.
No evidence was found that the C-derived body wall
muscle also depends on an induction, which suggests that
possibly three different pathways coexist in the early
embryo to specify body wall muscle, two of which are, in
different ways, influenced by cell-cell interactions and a
third that is autonomous.
This work supplies evidence that cells may acquire
transient states during embryogenesis that influence the
specification of other cells in the embryo. These states,
however, may not be reflected in the developmental potentials of the cells themselves. They can only be scored indirectly by their action on the specification of other cells in
the embryo.
Blastomeres that behave cell-autonomously in isolation
are nevertheless subjected to cell-cell interactions in the
embryonic context. Why this should be is an intriguing
question. The classical notion has been that blastomeres are
specified autonomously in nematodes. In recent years, it
was established that at least five inductions are required to
determine the AB descendants of C. elegans, whereas the
P1 descendants have been typically viewed to develop more
autonomously. It appears now that inductions also play a
major role during the determination of P1-derived blastomeres.
INTRODUCTION
expected from the fate map in isolation from the embryo, cells
are considered to be specified autonomously. Classical developmental biology introduced another criterion to distinguish
the state of specification of a primordium. When grafted to
ectopic positions in the embryo, only primordia that are not
reprogrammed to follow a new fate reflecting their new
position in the embryo are considered to be terminally determined. The biological meaning of this test, however, remains
obscure when the fate of cells can be changed even though they
are already restricted to their normal fate according to the fate
map (for review see Slack, 1991). Recently I showed, using
the early embryogenesis of C. elegans, that the experimentally
observed reprogramming of a primordium could be taken as an
indication that it is subjected to competing cell-cell interactions
in the embryo (Schnabel, 1994).
Two basic modes of cell determination are proposed for the
specification of cell fates during embryogenesis. In cellautonomous development, cells are instructed by intrinsic cues,
whereas in nonautonomous development cells depend on
signals from other cells to choose a certain pathway of differentiation. The classical experiment to determine the mode by
which a specific structure is specified is to isolate the primordium of the structure in question from the embryo to
prevent cell-cell interactions from occurring. If cells in
isolation do not acquire the fate expected from the fate map, it
is assumed that the normal fate of these cells is specified
nonautonomously by means of cell-cell interactions with other
cells. However, if the cells of the primordium execute the fate
Key words: cell specification, autonomy, nonautonomy, induction,
evolution, Caenorhabditis
2220 R. Schnabel
In vertebrates, the early specification pathway of most fates
requires interactions (for review see Slack, 1991). In invertebrates, especially in nematodes, some cell fates appear to be
specified cell-autonomously (Laufer et al., 1980; Cowan and
McIntosh, 1985; Edgar and McGhee, 1986; Schierenberg,
1988; Bowerman et al., 1992a; Mello et al., 1992). The body
of the nematode C. elegans is assembled by a stereotypic
lineage. During the first four cleavages of the fertilised zygote,
the stem cell like P cells (P0-P3) undergo asymmetric divisions
to produce the somatic founder cells AB, MS, E, C and D. The
blastomere AB produces hypodermis, neurons and pharynx.
MS produces pharynx and body wall muscle. E is the only
precursor of intestine. C gives rise to body wall muscle and
hypodermis. The blastomere D produces only muscle. The last
P cell, P4, is the precursor of the germ line (Sulston et al., 1983)
(Fig. 1).
The original notion was that nematodes develop strictly cellautonomously using determinants for the specification of cells
and tissues (zur Strassen, 1959). Recent evidence has shown
that the specification of the AB lineage depends on at least five
inductions (Priess and Thomson, 1987; Schnabel, 1991; Wood,
1991; Bowerman et al., 1992b; Hutter and Schnabel, 1994;
Mango et al., 1994; Mello et al., 1994; Moskowitz et al., 1994;
Hutter and Schnabel, 1995; Hutter and Schnabel, unpublished
data). There is experimental evidence that is consistent with
the notion that the other founder cells are specified cell
autonomously (Laufer et al., 1980; Cowan and McIntosh,
1985; Edgar and McGhee, 1986; Schierenberg, 1988;
Bowerman et al., 1992a; Mello et al., 1992). The potential to
form hypodermis, muscle and intestine is segregated from the
zygote P0 into the founder cells through the corresponding
lineages. The specification of intestine in E nevertheless
depends also on a cell-cell interaction required to polarise the
EMS blastomere (Schierenberg, 1987; Goldstein, 1992, 1993).
The MS, C and D blastomeres produce their normal tissues
when all other blastomeres present in the embryo are ablated
with a laser microbeam, which suggests, in accordance with
the classical criterion for cell autonomous specification, that
these tissues can be formed cell-autonomously (Laufer et al.,
1980; Cowan and McIntosh, 1985; Edgar and McGhee, 1986;
Bowerman et al., 1992a; Mello et al., 1992). However, it was
shown recently that one of the blastomeres, MS, despite its cell
autonomous potential to form muscle, is still subjected to counteracting interactions that are inhibiting and activating the production of body wall muscle from MS within the embryonic
context (Schnabel, 1994). It thus appears that blastomeres
execute their cell autonomous potentials when isolated but nevertheless are exposed, during normal development, to
inhibitory cell-cell interactions which in turn are overcome by
activating interactions. Here I describe a systematic search for
other competing, or duelling, cell-cell interactions in the early
embryo. By laser ablating different blastomeres or combinations of blastomeres, it is shown that the formation of body
wall muscle from D is inhibited by an interaction from the C
lineage which in turn is overcome by an activating signal from
the MS lineage. I further show that the inhibitory signal suppressing muscle specification described earlier for MS
(Schnabel, 1994) is derived from the D blastomere. This
inhibitory signal is transferred by the two descendants of C.
MATERIALS AND METHODS
Methods for culturing and handling of C. elegans have been described
by Brenner (1974) and Wood (1988). The standard wild-type strain
N2 is that of Brenner (1974). Laser ablation of blastomeres, immunostaining of irradiated embryos and the lineage analyses were carried
out as described previously (Schnabel, 1991, 1994; Hutter and
Schnabel, 1994). Laser operations of embryos were carried out at
25°C. The operated embryos were incubated at either 15°C for
approximately 9 hours, or at 20°C for approximately 6 hours or at
25°C for approximately 5 hours. The progress of embryogenesis was
monitored by observing the untreated embryos on the slides. All
treated embryos were scored for proper laser ablations just before
fixation. Properly irradiated blastomeres either did not divide at all or
divided only a few times. Embryos were processed for antibody
stainings when they reached the developmental stage shown in Fig.
2A corresponding to about 430 minutes of development at 20°C
(Sulston et al., 1983). Untreated embryos developed normally under
these conditions. All embryos were stained with mAb NE8 4C6.3
(antibody collection of the LMB MRC Cambridge England; Goh and
Bogaert, 1991; see also Schnabel, 1994).
RESULTS AND DISCUSSION
The C. elegans embryo produces 81 body wall muscle cells
derived from the AB (1), MS (28), C (32) and D (20) blastomeres (Fig. 1). The specification of the single body wall
muscle cell derived from AB is not considered further in this
work. By isolating the blastomeres C and P3, the mother of
D, I confirmed earlier work suggesting that body wall muscle
P0
AB
P1
0
ABa
ABp
10
20
EMS
P2
ABal ABar ABpl ABpr
MS
E
C
P3
30
40
50
60
D P4
70
80
hypodermis
germ line
nervous system
pharynx
hypodermis
pharynx
muscle 28
muscle 32 muscle 20
muscle 1
intestine
Fig. 1. The early embryonic lineage. During the first four cleavage
divisions of the zygote P0 the stem-cell like P cells produce, in
unequal divisions, five somatic founder cells and the germ line
precursor P4. The major tissues produced by the somatic lineages are
indicated below the lineages. The numbers of body wall muscle cells
contributed
by the different founder cells are indicated. The different
Fig.
1
lineages have different, characteristic cleavage rates. The time scale
indicated on the left corresponds to development in minutes at 25°C.
Duelling interactions in Caenorhabditis 2221
is produced cell autonomously from these blastomeres
(Cowan and McIntosh, 1985; Mello et al., 1992; Figs 2B,C,
3A).
As described earlier, the production of body wall muscle in
the MS lineage depends on an induction from the AB lineage
since an inhibitory signal emanating from the posterior P2
lineage must be overcome (Schnabel, 1994). During this work,
I often use the term lineage for convenience in a slightly
unusual way to avoid an ongoing repetition of many blastomere names. For example, the term MS lineage is used to
refer to the MS blastomere itself or its descendants or to several
generations of descendants. To search for further cell-cell
interactions required for the specification of tissues, embryos
of the appropriate stage were mounted on microscope slides
and blastomeres were ablated with a laser microbeam.
Developed embryos were evaluated by staining with the monoclonal antibody NE8 4C6.3 specific for body wall muscle (see
Schnabel, 1994) and by following the embryonic lineages of
embryos whose development was recorded with a 4-Dimensional Microscope (Fig. 4; Hird and White, 1993; see also
Fig. 2. Immunofluorescence micrographs showing body wall muscle
cell differentiation in normal and laser-ablated embryos. All embryos
are stained with mAb NE8 4C6.3. The two panels in each row except
for A, D and E show two different focal planes of each embryo.
(A1) Wild-type embryo at approximately 400 minutes of
development (20°C; Sulston et al., 1983) shortly after elongation has
started. Two of the four body wall muscle rows formed in the
embryo are seen. At this stage, the cytoplasm of the body wall
muscle cells is staining. The nuclei can be seen and counted. In
normal embryos, 81 body wall muscle cells stain. (A2) Wild-type
embryo at approximately 460 minutes of development. The muscle
cells start to produce filaments; however the nuclei can still be
identified. Soon after this stage, the general cytoplasmic staining
disappears as the sarcomere develops and it is impossible to count
the number of body wall muscle cells. Therefore manipulated
embryos were evaluated only when the cytoplasmic staining was still
present. Shortly before hatching the antibody stains faintly at least
four more muscles which are not body wall muscles. These muscles
are therefore not considered further in this work. (B-I) Examples of
laser ablated embryos from the experimental series shown in Fig. 3.
(B) Embryo in which all cells except for P3, the mother of D, were
ablated. A total of 20 muscle cells were observed in this embryo.
This is the equivalent normally produced by D. (C) Embryo in which
all cells except for C were ablated. I counted 31 muscle cells in this
embryo. C normally produces 32 muscle cells. (D) An embryo with
an ablated EMS blastomere. I counted 31 muscle cells in this
embryo. If C and D would produce their full complements of muscle
this embryo should contain 53 (AB 1, C 32, D 20) muscle cells.
(E) An embryo where EMS and C were ablated. The number of
muscle cells observed in this embryo was 20, the number normally
produced by D. (F) Embryo with ablated ABa and C blastomeres. A
total of 34 muscle cells were observed in this embryo. Therefore MS
should have produced 14 muscle cells in the embryo. (G, H)
Embryos where ABa and D were ablated at the cleavage from two to
four C descendants (G) or about 10 minutes later when the two E
descendants cleave (H). The earlier D ablation still partially relieves
the inhibition of MS-derived muscles. I counted 48 muscle cells in
this embryo (G). When D is ablated later (H) the MS muscle is
already fully suppressed. I counted 33 muscle cells in this embryo.
(I) An embryo where first EMS was ablated and then later the two C
descendants Ca and Cp were ablated just before the onset of their
mitoses. Most of the inhibition of D already occurred in this embryo,
only 6 muscle cells could be detected. Bar 10 µm.
Schnabel, 1991; Hutter and Schnabel, 1994). It was shown
before that laser ablations of individual blastomeres do not
affect the remainder of the embryo unspecifically (Schnabel,
1994; Hutter and Schnabel, 1994).
A1
A2
B1
B2
C1
C2
D
E
F1
F2
G1
G2
H1
H2
I1
I2
2222 R. Schnabel
A) Isolated C and D blastomeres
produce muscle autonomously
1)
2)
ABp
3)
ABp
ABa
P3
EMS
EMS
AB MS C D L 26 H 32
81 79 ± 2 (5)
∆~2
Count of muscle
cells in untreated
embryos
ABa
P3
Autonomous
muscle
development in
the C lineage
Autonomous
muscle
development in
the D lineage
Approximately Ablation of EMS and
20 muscle cells P3 has no further effect
missing. Those than just ablating EMS
from D?
C) The interaction between the EMS and the D lineage occurs
before the onset of gastrulation
1)
2)
3)
4)
8 AB
ABp
ABa
P2
EMS
MS
8 AB C
C
P3
E
53 31 ± 2 (10)
∆ ~ 22
D muscles missing
see also Fig. 4
41 ± 2 (6)
∆ ~ 12
53
MS
7 ± 2 (3)
∆ ~ 14
21
Ablation of C relieves
the inhibition of
muscle production in
D
After a late ablation of
the 2C blastomeres
most muscles from D
are already inhibited
D) ABa overcomes an inhibition of muscle in MS by the P2
and ABp lineages ((1 to 3) from Schnabel, 1994)
3)
1)
2)
C
ABp
C
C
MS MS
ABa
P2
ABa
P2
EMS
P4
ABp
ABp
ABa
D
E E
MS
P2
EMS
EMS
AB MS C D L 48 H 52
AB MS C D L 47 H 54
AB MS C D L 10 H 18
AB MS C D L 25 H 28
50 ± 1 (7)
∆~3
81 49 ± 2 (11)
∆ ~ 32
29 15 ± 3 (7)
∆ ~ 14
28 25 ± 3 (6)
∆~3
53
D derived muscle
partially expressed
D muscles missing
see also Fig. 4
32 AB C
P4
AB MS C D L 39 H 44
32 ± 2 (6)
∆ ~ 21
53
E
MS MS
E
AB MS C D L 28 H 33
AB MS C D L 29 H 36
C D
D
P4
AB MS C D L 6 H 9
20 ± 1 (5)
∆~1
21
C
C
EMS
AB MS C D L 19 H 21
30 ± 2 (7)
∆~3
33
ABp
ABa
P3
EMS
AB MS C D L 28 H 33
53 31 ± 2 (10)
∆ ~ 22
4)
C
ABa
P3
EMS
AB MS C D L 29 H 36
18 ± 2 (5)
∆~2
20
ABp
C
ABa
P2
EMS
AB MS C D L 16 H 20
30 ± 2 (6)
∆~2
32
ABp
ABp
C
ABa
EMS
AB MS C D L 76 H 81
ABp
C
ABa
P2
B) The EMS lineage is required to overcome an inhibition of
muscle production in the D lineage by the C lineage
2)
3)
1)
MS-derived
muscle inhibited
D derived muscle
expressed
Inhibition
partially removed
Inhibition
completely removed
D) continued, the blastomere D inhibits muscle production in MS. The inhibitory signal derived from ABp is induced by the ablation of P2
4)
5)
ABp
ABp
C
ABa
ABa
P3
EMS
61
60± 2 (7)
∆~1
An early ablation of
P3 relieves muscle
production in MS
D) continued
10)
ABp
D
ABa
C
E
MS MS E
61
60
C C
ABa
54 ± 5 (6)
∆~7
Ablation of D right
after its birth relieves
most of the inhibition
of muscle in MS
MS
MS
MS MS E
P4
E
AB MS C D L 36 H 46
61
40 ± 4 (6)
∆ ~ 21
E) Some further ablations one might think of
1)
2)
ABp
ABa
P2
AB MS C D L 0 H 1
1
0 ± 1 (3)
∆~1
No supernumerary
muscles produced in
the AB lineage
ABa
The MS derived muscle is
already strongly inhibited
when D and P4 are ablated
at the 2 to 4 C cell cleavage
ABa
P2
ABp
C D
C
P4
E
MS MS E
ABa
P4
AB MS C D L 28 H 33
61
AB MS C D L 50 H 53
32 ± 2 (9)
∆ ~ 29
81
The inhibition of MS derived
muscle is completed when D
and P4 are ablated at or just
after the 2 to 4 E cell cleavage
51 ± 1 (5)
∆ ~ 30
Ablation of P4 right
after its birth does not
relieve the inhibition of
muscle in MS
Lowest and highest
muscle cell count in
individual embryos of
the series
Blastomere ablated
with laser
AB MS C D L 31 H 33
32 ± 1 (5)
∆ ~ 29
The inhibition of MS derived
muscle is completed when D is
ablated at or just after the 2 to
4 E cell cleavage
3)
2)
ABp
EMS
E
41 ± 5 (6)
∆ ~ 20
4C
4E
4MS P4 D
ABp
EMS
61
ABa
P4
D
ABp
61
MS derived muscle is
already strongly inhibited
when D is ablated at the 2
to 4 C cell cleavage
MS
MS
MS MS E
9)
4C
4E
4MS
ABp
KEY
12)
D
8)
D
AB MS C D L 32 H 45
59 ± 2 (4)
∆~1
The ABp lineage does
not inhibit muscle in
MS after an early
ablation of P3
ABp
P4
AB MS C D L 48 H 59
61
AB MS C D L 58 H 62
A late ablation of
P3 partially
relieves muscle
production in MS
ABa
P3
EMS
55± 7 (6)
∆~6
ABp
C
AB MS C D L 44 H 62
11)
C
ABa
P3
EMS
AB MS C D L58 H 60
ABp
C
C C
7)
6)
ABa
P2
EMS
AB MS C D L 30 H 31
AB MS C D L 28 H 32
53 31 ± 1 (3)
∆ ~ 22
52 31 ± 2 (5)
∆ ~ 21
ABa is not involved
in the inhibition of
muscle in D
ABp is not involved
in the inhibition of
muscle in D
Cells contributing
to muscle
Number of
muscle cells
contributed
ABp
ABa
P2
EMS
AB MS C D
1 28 32 20
81
Expected number of
cells if specification is
cell autonomous
L 47 H 54
49 ± 2 (11)
Number of
embryos
∆ ~ 32
Number of counted cells
with standard deviation
Muscle contribution removed
by laser ablation
Difference between the
observed number of cells
and the number of cells
expected for an
autonomous specification
AB MS C D
Duelling interactions in Caenorhabditis 2223
Specification of body wall muscle in D
If the specification of the 52 body wall muscle cells derived
from the blastomeres C and D occurred only cell
autonomously, then the ablation of any other blastomere in the
early embryo should not interfere with muscle development in
these lineages. The ablation of ABa or ABp in the 4-cell-stage
embryo has indeed no effect on the specification of muscle in
either C or D (Schnabel, 1994). However, after ablation of the
EMS blastomere muscle development is affected. If muscles
were specified cell autonomously in the C and D lineages, one
would expect to find 53 (C 32, D 20, AB 1) instead of the
normal 81 muscle cells since MS, the daughter of EMS, contributes 28 cells to the body wall muscle. However, after the
ablation of EMS the number is reduced to 31±2 (± s. d.) muscle
cells (Figs 2D, 3B). The observed reduction of body wall
muscles indicates that either the C or D or even both lineages
require an induction from the EMS lineage to produce body
wall muscle. Since the number of missing muscle cells is very
close to the number produced by D (20), I tested by a double
ablation of EMS and P3, the mother of D, whether indeed the
Fig. 3. Interactions modulating body wall muscle specification in the
early embryo (key to figure in lower right panel). (A1) Muscle
development in untreated embryos. (A2) An isolated C blastomere
produces body wall muscle cell-autonomously. (A3) After isolation
of P3, the D blastomere produces body wall muscle cellautonomously. (B) Within the embryonic context, the EMS lineage is
required to overcome an inhibition of muscle production conferred
by the C lineage. After the ablation of EMS (B1), approximately 20
muscle cells are missing. (B2) The additional ablation of P3 does not
affect muscle specification further. (B3) If C is ablated in addition to
EMS, the inhibition of body wall muscle in D is relieved, which
suggests that the C lineage is suppressing muscle specification in the
D lineage. (B4) The two C descendants Ca and Cp are the inhibitors.
After a late ablation of these blastomeres, most muscle derived from
D is already suppressed. (C) Timing of the activation of muscle in
the D lineage. (C2) Ablation of MS still fully inhibits muscle
production in D. After ablation of the two descendants MSa and
MSp (C3), the activation occurred partially; after the ablation of the
four descendants of MS, at the onset of gastrulation the activation
occurred fully (C4). (D) The blastomere D is the inhibitor of body
wall muscle production in the MS lineage and the ABa lineage is
required to overcome this inhibition. (D1-3) Again the initial set of
experiments demonstrating that muscle formation in MS is exposed
to duelling interactions (Schnabel, 1994) to facilitate the
understanding of the following experiments. (D4-12) Experiments
determining the timing of the inhibition and identifying the
blastomere D derived from the P2 lineage (Fig. 1) as the blastomere
inhibiting muscle production in MS. An early ablation (D4) of the P3
blastomere relieves the inhibition of the MS lineage completely.
After a late ablation of this blastomere some muscles are already
inhibited (D5). (D7,8) The ablation of the two P3 descendants D and
P4 at the time when Ca and Cp divide relieves the inhibition of MS
muscles partially, after the two E descendants have divided complete
relief is observed. Ablation of only P4 does not (D9), ablation of D,
however, does (D10) relieve the inhibition of MS-derived muscles,
which indicates that the D lineage is the source of the inhibitory
signal. (D11,12) The ablation of D at the time when the two C
descendants Ca and Cp divide relieves the inhibition of MS muscles
partially; after the two E descendants have divided complete relief is
observed. (E) Some further ablations. (E1) The AB lineage does not
produce supernumerary body wall muscle cells in isolation. (E2,3)
Neither the ABa nor the ABp lineage are involved in the inhibition
of muscle in the D lineage.
contribution of D is missing. The rationale of this experiment
is that the additional ablation of a blastomere that is unable to
produce muscles due to a missing interaction should not make
any difference. If, however, the missing muscles were
normally produced by a cell different from the ablated one the
number of muscle cells counted after this ablation should be
further decreased. The number of muscle cells counted after
the double ablation is the same (30±2) as when only EMS is
ablated (31±2) (Fig. 3B). I took this as evidence that it is indeed
the body wall muscle derived from D that is missing in EMS
ablated embryos. This was confirmed by directly observing the
muscle differentiation using the 4-Dimensional Microscope. In
embryos where either EMS or MS were ablated, prospective
muscle cells in the D lineage underwent an additional round of
mitoses, which suggests that these cells are not specified
properly (Fig. 4). Muscle cells derived from C, which are not
affected according to the immunochemical analyses, differentiated normally into body wall muscles when observed directly
using the 4-Dimensional Microscope.
The D lineage is subjected to an inhibitory signal
The result concerning muscle specification in D raises the same
dilemma observed earlier when studying the muscle specification in the MS lineage. The isolated founder cell can produce
its tissue apparently autonomously; however, it is possible to
demonstrate that the specification of this same tissue within the
embryonic context requires an induction. This dilemma can
only be resolved if an inhibitory interaction normally suppresses the autonomous tissue specification (Schnabel, 1994).
In this situation, the repression of muscle cell specification
should be relieved if the activating and inhibiting signals are
both removed. This is what also happens in an isolation experiment. The inductions here referred to as ‘activating’ are
indeed, as will be apparent later, ‘counter-inhibitory’ interactions which serve to overcome other inhibitory interactions.
For the matter of simplicity, the counter-inhibitory signals are
nevertheless referred to as activating signals throughout the
manuscript because, in the presence of the inhibition, the
experimental effect observed is an activation.
Concerning the inhibition of muscle in the D lineage, I could
show that, after ablation of EMS and C, the D lineage produces
its normal equivalent of 20 (20±1) muscle cells (Figs 2E, 3B),
which suggests that the C lineage normally suppresses muscle
development in D. The role of the C lineage in suppressing
body wall muscle in D is discussed later in more detail. EMS
is required to overcome this inhibition. Double ablations of
either ABa or ABp and EMS do not relieve the suppression of
muscle production from D (Fig. 3E).
These results indicate that the MS lineage is required for the
proper specification of the D lineage, which is derived from
the P2 lineage. I earlier reported that the production of body
wall muscle in the MS lineage itself is inhibited by the P2
lineage (Schnabel, 1994). Therefore the interactions between
these two lineages are reciprocal. The P2 lineage suppresses
muscle specification in the MS lineage whereas the MS lineage
is required to permit muscle specification in the blastomere D
which is derived from the P2 lineage.
The timing of the interactions
It appears that a complex network of duelling interactions
exists in the early C. elegans embryo. ABa is required for
2224 R. Schnabel
C
D
Normal
A
ABp
Lineage could not be determined
EMS
ABa
P2
EMS
? ??
?
EMS
B
8 AB
C
MS
P3
MS E
? ?
MS
C
8 AB
MS E
C
Lineage ablated
Lineage ablated
MS + P3
P3
MS + P3
MSap
MSpp
D
Normal
D
ABp
ABa
ABa
P2
EMS
??
??
ABa
?
E
ABp
ABa
EMS
C
ABa + C
*
P3
Mitosis
Muscle
? Lineage not resolved
Cell death
Hypodermis
Large round cell
Fig. 4. Lineage analyses of ablated embryos. The upper part of the figure depicts the C and D lineages, the lower part MS-derived lineages
producing body wall muscle and again the D lineage. The C lineage produces mostly hypodermis and muscles, the MS lineage produces
pharynx (not shown) and body wall muscles. The D lineage produces only muscles. The figure shows the results of lineage analyses using
the 4-D microscope. After the ablation of either EMS (A) or MS (B), the C-derived muscles develop normally as far as this can be judged
using the microscope. Muscle precursors show the typical migration pattern and assemble into rows. However, the D-derived muscle cells
undergo additional mitoses, an indication that these cells are not specified properly. After initially migrating with the other muscle
precursors, cells often leave the muscle rows later and then undergo the additional mitosis. The C-derived hypodermal cells mostly
developed normally. For a few cells I observed an additional mitosis. The evaluation of the hypodermal lineages proved to be very difficult
in these embryos, which may indicate that these cells were somehow not completely normal. The significance of these additional mitoses
remains to be clarified. (C) The lineage of the C-derived hypodermis was determined in two embryos where MS and P3 were ablated. I
easily scored the hypodermal cells to be normal in these embryos. A normal differentiation of the hypodermis should be expected in these
embryos if the P3 lineage not only acts as an inhibitor of body wall muscle differentiation in the MS lineage but also influences hypodermal
differentiation in the C lineage which transfers the signal inhibiting muscle in the MS lineage. (D) Lineage analysis of ABa ablated
embryos. MS-derived muscles either undergo an additional mitosis or differentiate into large round cells which somehow resemble
hypodermal cells in these embryos. This confirms my earlier conclusion that the expression of body wall muscle depends on an induction
from the ABa lineage (Schnabel, 1994). The D lineage produced body wall muscles in these embryos. (E) The ablation of C partially
relieves the inhibition of MS-derived body wall muscles in ABa ablated embryos. The immunochemical analysis suggests that the MS
lineage produces approximately 14 muscle cells in doubly ablated embryos. This is reflected in the terminal differentiation of the analysed
lineages normally producing muscle in the MS lineage. Approximately half of the cells differentiated aberrantly. (*) The cell MSapppp
arrested one cleavage too early and developed into a large cell with hypodermal appearance. The D lineage produced body wall muscles in
this embryo.
Duelling interactions in Caenorhabditis 2225
Inhibition of muscle in the MS lineage
In my previous work I showed, using immunochemical
methods, that the specification of body wall muscle in the MS
lineage requires a signal from the ABa lineage to counteract
an inhibitory signal derived from the P2 lineage (Schnabel,
1994). The conclusion that it is the MS-derived body wall
muscle that requires the signal from the ABa lineage was now
confirmed by following MS lineages producing body wall
muscle in the 4-Dimensional Microscope (Fig. 4D). To identify
the P2-derived blastomere which produces the inhibitory signal
and to determine the timing of the inhibitory signal, I first
ablated ABa in embryos to remove the signal activating muscle
in MS. To identify the source of the inhibitory signal which
now can be scored in absence of the activating signal, I ablated
additionally P3 very early or late in its cell cycle or both of its
daughters, either around the time when the two C blastomeres
are cleaving, or after the cleavage of the two E cells (Figs 1,
3D). An early ablation of P3 still fully relieves the inhibition
of body wall muscles in the MS lineage. The consequences of
the ablation of C, the other daughter of P2, will be discussed
below in a different context. If both daughters of P3, P4 and
D are ablated at the time when the two C descendants cleave,
most of the body wall muscles are already suppressed (approximately 70%). Ablation of both blastomeres right at or after the
cleavage of the two E descendants about 10 minutes later
shows that the inhibition is already completed at that time of
development (Fig. 3D). To identify which of the daughters of
P3, D or P4 is producing the inhibitory signal, I ablated ABa
and P4 or D. Ablation of P4 does not relieve the inhibition sug-
100
P2
0
8 ABa 4MS
P3 C
80
P3
20
D
4 ABa
40
60
40
2MS
60
2C
D
2 ABa
20
ABa
EMS
80
P4
MS
% body wall muscles inhibited
Activation of muscle in the D lineage
To determine the timing of the induction of muscle in the D
lineage, I ablated EMS or the blastomeres of the MS lineage
at different stages of early development. In these experiments,
the inhibiting signal derived from the C lineage remains intact,
but the source of the activating signal is removed at different
times of development. If the activation did not yet occur, the
D-derived muscle should still be suppressed. If the ablation of
the source is carried out after the activation occurred D should
express its normal equivalent of body wall muscles. Ablation
of EMS or MS completely suppresses muscle formation in the
D lineage. After the ablation of the two MS descendants 40%
of the D-derived muscles are formed, an indication that a
partial activation already occurred. If the 4 MS descendants are
ablated soon after their birth, the full equivalent of D-derived
muscles is formed, which shows that the activation is already
completed. I therefore conclude that the activation of body wall
muscle in the D lineage occurs late at the 2 MS cell stage
around the time when the D blastomere is born (Figs 3C, 5).
gesting that the D lineage is the source of the inhibitory signal.
Indeed, the ablation of the D blastomere right after its birth
relieves most of the inhibition, approximately 75% of the MSderived muscles are produced. If D is ablated 10 minutes later
% body wall muscles produced
muscle production in MS, which is repressed by the P2 lineage.
MS itself is required to overcome an inhibitory signal within
the P2 lineage. Two formal scenarios could explain the
complex interactions. (i) There exist discrete independent
interactions among the blastomeres which could be required at
different times of development. (ii) Alternatively, the complex
interaction pattern could be generated by a network of reciprocal interactions occurring simultaneously. To distinguish
between these two possibilities I determined the timing of the
inhibitory and activating signals, which are discussed in the
following three sections.
D
100
0
Window of
interactions
EMS
MS
C
P2
P3
2MS
4MS
4C
2C
D
P4
10 minutes
at 25 °C
12- cell stage
24- cell stage
Fig. 5. Timing of the interactions modifying muscle development in
the MS and D lineages. The upper part of the figure shows the
inhibition or activation of body wall muscle cells when different
blastomeres are ablated at the different embryonic stages indicated in
the lower part of the figure. The horizontal bars indicate the times of
existence of the different blastomeres. The triangles correspond to
the activation of body wall muscle in the MS lineage by the ABa
lineage (data from Schnabel, 1994). The activation occurs around the
12-cell stage of the embryo. The circles indicate the activation of the
D-derived body wall muscle by the MS lineage (data from Fig. 3C).
The activation occurs around the late 24- / early 26-cell stage and
thus one cleavage later than the activation of MS. The squares
indicate the inhibition of muscle production in the MS lineage by the
P2 lineage and its descendants after the activating ABa lineage was
ablated (data from Fig. 3D). The inhibition occurs exactly at the
same time as the reciprocal activation of the D lineage by the MS
lineage. The two crosses indicate the timing of the inhibition of the
D-derived muscle by the C lineage. After an ablation of the
activating MS lineage the ablation of the blastomere C still fully
relieves the inhibition of D. When the two C descendants Ca and Cp
are ablated shortly before they undergo mitoses most of the muscle
derived from D is already inhibited. Therefore the inhibition of D
appears to follow the same time course as the other interactions in
the posterior of the embryo.
2226 R. Schnabel
together ABp never elucidates an inhibitory activity (Figs 3D4
and 6A3-5).
at the cleavage of the two C descendants most of the inhibition of MS-derived muscles is observed and only approximately 35% of the muscles are produced in the MS lineage.
The MS-derived muscle is fully suppressed when D is ablated
another 10 minutes later at or after the cleavage of the two E
descendants. These results suggest that the D blastomere itself
is the source of the inhibitory signal suppressing muscle in the
MS lineage (Figs 2G,H, 3D).
I reported earlier that the ABp lineage also functions as an
inhibitor if ABa and P2 are ablated (Schnabel, 1994). The
results presented here show that this is very probably due to
the P2 ablation itself. When ABa and additionally the
daughters of P2, P3 and C are either ablated on their own or
Simultaneous exchange of signals among
blastomeres
A graph showing the time courses of the interactions is
depicted in Fig. 5. The curves corresponding to the activation
of the muscle specification in the D lineage and to the inhibition of muscle specification in the MS lineage cross at the late
24-cell / early 26-cell stage of the embryo. This is the time
when the blastomere D is born. The time window in which the
interactions occur corresponds to approximately 30 minutes at
25°C. Within the inherent limit of the resolution of the laser
A) The inhibitory signal derived from D suppressing body wall muscle in MS is transferred through the C lineage
1)
ABp
ABa
ABa
P2
EMS
EMS
ABp
C
ABa
P3
EMS
AB MS C D L 49 H 53
AB MS C D L58 H 60
81 51 ± 2 (5)
∆ ~ 30
61
Ablation of ABa inhibits
the MS derived muscles
(Schnabel, 1994), new
embryos from this work
A) continued
ABa
P3
EMS
49
An early ablation of
P3 relieves muscle
production in MS,
taken from Fig. 3D4
C
35 ± 3 (6)
∆ ~ 14
48
Ablation of C
partially relieves the
inhibition of MS
derived muscles
ABa
P3
AB MS C D L 29 H 38
AB MS C D L 31 H 38
60± 2 (7)
∆~1
ABp
34 ± 4 (6)
∆ ~ 14
ABp does not act as an
inhibitor after ablation of
C (compare Fig. 3D1- 6)
EMS
ABp
C
P3
AB MS C D L 18 H 28
29
ABa
24 ± 4 (5)
∆~5
The P3 lineage is the
initial source of the
inhibitory signal
(compare Fig. 3D5)
Cp
Ca
EMS P3
AB MS C D L 42 H 56
65
49 ± 5 (6)
∆ ~ 16
Ablation of Cp
partially relieves the
inhibition of muscles
derived from MS
B) The MS derived signal activating body wall muscle in D inhibits the potential of the
C lineage to suppress muscle in D
4)
5)
2)
1)
3)
7)
ABp
ABp
ABa
ABp
C
6)
5)
4)
3)
2)
ABp
P2
MS E
AB MS C D L 49 H 58
81 52 ± 3 (8)
∆~1
Ablation of E has no
effect on the inhibition
of MS derived muscles
ABa
ABp
ABa
P2
EMS
AB MS C D L 29 H 36
53 31 ± 2 (10)
∆ ~ 22
EMS
ABa
P3
AB MS C D L 31 H37
37
ABp
Cp
Ca
36 ± 1 (4)
∆~1
EMS
ABa
P3
AB MS C D L 63 H 66
65
ABp
Cp
Ca
65 ± 1 (6)
∆~0
Ablation of only Cp
After ablation of EMS Ablation of Cp
the D derived muscle removes the inhibition has no effect on the D
of D derived muscles derived muscles
is missing (see Figs.
3B and 4)
Cp
Ca
EMS
P3
AB MS C D L 44 H 51
65
48 ± 3 (6)
∆ ~ 17
ABp
ABa
MS
MS Ea Ep
P2
AB MS C D L 79 H 81
81 80 ± 1 (5)
∆~1
Ablation of only Ca
Ablation of Ea has
suppresses the muscle no effect on muscle
derived from D
specification
Fig. 6. The reciprocal interactions between the MS and D lineages are mediated by the C lineage. (A) The inhibitory signal suppressing body
wall muscle formation in the MS lineage derived from the blastomere D is transferred by the descendants of the C blastomere. In all embryos,
the ABa blastomere was ablated to remove the signal activating muscle in the MS lineage in order to permit the detection of the inhibitory
signal derived from the blastomere D. (A1,2) As shown before in Fig. 3D, the ablation of the blastomere P3 relieves the inhibition of muscle in
the MS lineage completely. Neither the ABp nor the C lineage act as inhibitors in this situation. (A3) Nevertheless the suppression of body wall
muscle is relieved when the C lineage is ablated, which suggests that it is required to transfer the inhibitory signal. (A4,5) The ABp lineage
does not act as an inhibitor when only C or C and P3 are ablated. This experiment excludes the possibility that ablation of C fully relieves the
inhibition of the MS lineage but in turn body wall muscle is suppressed by the ABp lineage as with the ablation of P2. (A5) Late ablation of C
and P3 removes the inhibition of muscle in the MS lineage to the same extent as ablating only P3 at the same time (Fig. 3D5). This suggests
that the P3 lineage, that is the blastomere D, is the initial source of the signal whose transfer is hampered by the ablation of C. The experiment
also provides evidence that it is the ablation of P2 itself that causes the ABp lineage to inhibit muscle in the MS lineage. (A6) The ablation of
only the blastomere Cp has the same effect as the ablation of the whole C lineage. This corroborates the argument that the C lineage on its own
does not inhibit muscle in the MS lineage since Ca is normal in this experiment and could still function as an inhibitor. Cp is located
completely sideways from the direct line between the D blastomere and the MS descendants. This makes it rather probable that the inhibitory
signal is really transferred either directly through or over the surface of the C descendants. It is rather improbable that the ablation of Cp should
interfere with a free diffusion of a soluble factor between D and MS descendants. (A7) The ablation of the E lineage whose descendants span,
like the descendants of C, the gap between the descendants of MS and the blastomere D has no effect, which suggests that this lineage is not
involved in the transfer of a signal. (B) The MS-derived signal activating body wall muscle in the D lineage acts indirectly by inhibiting the
inhibitory function of the C lineage. (B1,2) The ablation of Cp relieves the inhibition of muscle formation from D observed after the ablation of
only EMS. (B3) The ablation of only Cp has, as expected for the removal of an inhibitor, no effect on D-derived muscles. (B4) The ablation of
only Ca, however, blocks the differentiation of body wall muscles in the D lineage. This indicates that the MS lineage does not activate the D
lineage directly and that the C lineage is required to transfer the signal (B5). In contrast the ablation of Ea has no effect on muscle specification.
Duelling interactions in Caenorhabditis 2227
ablation experiments (it may take a few minutes after the
ablation is finished until a blastomere is inactivated), it appears
that the relation between the MS and D lineage is reciprocal.
The activating and inhibiting signals are exchanged simultaneously at about the time when the D blastomere is born.
To address the question whether the inhibition of body wall
muscle production in the D lineage by the C lineage also occurs
at the same time as the interactions between the MS and D
lineages, I ablated EMS to remove the activation of muscle in
D and then ablated the two C descendants Ca and Cp very
shortly before they cleave again. As already described, the
ablation of C itself completely relieves the inhibition of muscle
in the D lineage. If the inhibition of body wall muscle in the
D lineage by the C lineage would follow the same time course
as the inhibition of body wall muscle in the MS lineage, one
would expect that only about 30% of the D-derived body wall
muscles should be formed when Ca and Cp are ablated shortly
before their division. After ablation of these cells indeed
approximately 7 instead of the normal 20 body wall muscles
derived from D are produced. This corresponds to approximately 30% of the D-derived muscles. Therefore, it is very
probable that the interaction between the C and D lineage
occurs at the same time as the other interactions (Figs 3B, 5).
The D-derived signal suppressing muscle in the MS
lineage is transmitted by the C lineage
The signals could be either exchanged by long-range diffusible
signals or they could be transferred through cell-cell contacts
among blastomeres. Since very short-range diffusible signals
cannot be distinguished with the methods used here from
signals transferred by direct cell-cell contacts, these two mechanisms are not distinguished further in this work. Cell-cell
contacts confer the specificity of the inductions specifying the
AB lineage (Hutter and Schnabel, 1994; 1995; Moskowitz et
al., 1995). To discriminate between the two possibilities for the
inhibition of the MS lineage by D, I carried out double
ablations ablating ABa to remove the signal activating muscle
in MS, and C or E whose descendants could themselves be part
of a signal chain between D and the MS descendants (the
topography and the cell-cell contacts in 24-cell embryos are
discussed below; Fig. 7). Ablation of E does not interfere with
the inhibition of body wall muscle in MS by the D lineage. The
ablation of C, however, partially relieves the inhibition of
muscle production in MS. The immunochemical analysis of
body wall muscle specification in these embryos suggests that
approximately 14 body wall muscles are produced by the MS
lineage (Figs 2F, 6A3-5). A lineage analysis shows that the MS
lineage produces indeed some body wall muscles when C is
ablated in addition to ABa. The D lineage develops normally
after the ablation of ABa and C (Fig. 4E). The C lineage thus
acts formally as an inhibitor of muscle production in the MS
lineage. However, the C lineage depends completely on the P3
(D) lineage to acquire its inhibitory function. Ablation of P3
alone, which leaves the C lineage intact, completely relieves
the inhibition of the MS lineage (Fig. 3D4). The C lineage on
its own thus does not inhibit the MS lineage. Therefore I
propose that the inhibitory signal from D is transferred through
the C lineage. To find further evidence that the inhibitory signal
is transferred through C descendants I ablated the MS activating lineage ABa and the blastomere Cp immediately after its
birth at a time when the inhibitory blastomere D is not yet born.
A
Ca
Cp
D
MSa
B
MSp
C
MSa
MSp
Ca
Cp
Fig. 7. Cell-cell contacts in a late 24-cell embryo. (A,B) Nomarski
micrographs. Left ventral views. (A) Top optical section.
(B) Bottom section. The D blastomere always touches the
blastomere Cp. The C-derived and MS-derived blastomeres lie on
the dorsal and ventral side of the embryo, respectively. They only
touch in the inner of the embryo. (C) The contact between MSp
and Ca can be seen in an electron micrograph kindly provided by
T. Cole. Bars 10 µm.
2228 R. Schnabel
During this experiment, Ca, which touches the MS descendants
(Fig. 6), stays intact and could thus inhibit the MS lineage on
its own. The ablated blastomere Cp, however, ‘isolates’ Ca
from P3 and D, respectively. After this manipulation, the inhibition of body wall muscle production from MS is relieved to
the same extent (approximately 50%) as when the mother of
these cells, C, is ablated (Fig. 6). This indicates that Ca does
not inhibit the MS lineage on its own, but that the D lineage is
required for the inhibitory activity. It appears that Ca depends
on Cp to execute an inhibitory function or to transmit an
inhibitory signal.
I have shown previously that the ABp descendants, which
are also located in the posterior of the embryo, do not have a
role in propagating the D-derived signal. After ablation of the
entire AB lineage, which removes the activating signal from
ABa but leaves EMS, C and P3 (D) intact, the MS-derived
muscle is fully suppressed and the C- and D-derived muscles
are expressed normally (Schnabel, 1994).
The MS-derived signal activating muscle in the D
lineage is also transmitted by the C lineage
As discussed above, the C lineage acts as an inhibitor of muscle
development in the D lineage. This inhibition is overridden by
the MS lineage. Again, I wanted to examine by which path the
activating signal is transmitted. ABp-derived blastomeres
which are located in the posterior of the embryo do not appear
to have a role in the transfer of the signal as the ablation of
ABp alone has no influence on the transfer of the activation to
D (Schnabel, 1994). I directly wanted to test the question of
whether the signal is also transferred by the C lineage. This
question, however, leads to the experimental dilemma that the
ablation of the C lineage, which possibly transmits the signal,
also removes the source of the inhibition, which is derived
from the same lineage. It was shown earlier that the ablation
of C fully relieves the inhibition of D (Fig. 3B3). However, if
the two descendants of C, Ca and Cp are ablated very late
shortly before their division, most of the D-derived muscle is
already inhibited (Fig. 3B4).This suggests that both or one of
the descendants function as an inhibitor. The fact that the
inhibitory signal is only sent after the two C descendants are
born permits to design two experiments to test whether the activating signal is also transmitted through the C descendants.
The first experiment is to ablate EMS and Cp. Ablation of EMS
removes the activation of the D-derived muscle, which allows
testing of whether Cp, which touches D (Fig. 7), can act as an
inhibitor by itself. Indeed, an additional early ablation of Cp
relieves the inhibition of the D-derived muscle (Fig. 6B2),
which suggests that the Cp lineage inhibits the muscle in D.
As expected for an inhibitor, ablation of only Cp has no effect
on D (Fig. 6B3). If Cp on its own acts as an inhibitor, it is
possible to test whether the activating signal from the MS
lineage is transferred by the C descendants or by the remainder
of the embryo by ablating only Ca. Ablation of only Ca leaves
both the activating lineage MS and the inhibiting lineage Cp
intact. If the activating signal is transmitted by a path different
from the C descendants to D, the ablation of only Ca should
have no effect. If, however, the activating signal passes
through the C descendants, the ablation of Ca alone should
block the transmission of the signal which in turn should
permit Cp to suppress muscle in D. As shown in Fig. 6B4, the
ablation of only Ca suppresses the D-derived muscle. As a
control, I also ablated only Ea which had no effect (Fig. 6B5).
These results suggest that both the activation and the inhibition of the D-derived muscle occur via the C lineage. As will
be discussed later, MS could possibly directly influence the C
lineage and thus relieve the inhibition of the D lineage.
Cell-cell contacts among the interacting
blastomeres
The presented experiments supply evidence that the MS
lineage interacts with the D lineage, the D lineage with the MS
lineage and the C with the D lineage. The reciprocal interactions between MS and D depend on the C descendants, which
suggests that the signals do not diffuse freely in the posterior
of the embryo but are transferred directly through the C
descendants. This raises the possibility that the signals are
passed through direct contacts among blastomeres. To see
whether indeed direct cell-cell contacts exist between the interacting blastomeres, I analysed the cell-cell contacts in 24-cell
embryos, the stage when the interactions occur. Cell-cell
contacts between the descendants of D and C are obvious in
embryos inspected under the light microscope (Fig. 7). The two
MS and two C descendants lie, however, on opposite sides of
the embryo; therefore, MSp and Ca could only have contact in
the centre of the embryo. This contact can be seen in the light
microscope but is very hard to depict in pictures. In an electron
microscopic section of a 24-cell-stage embryo, published
already by Krieg et al. (1978), the contact between MSp and
Ca can clearly be seen (also shown in Fig. 7C; the picture was
kindly provided by T. Cole). Thus interactions among the
lineages MS and C and C and D, respectively, could occur
through direct cell-cell contacts.
Is the C lineage itself influenced by the interactions?
None of the described ablation experiments had an effect on
the body wall muscle specification in the C lineage. Thus, body
wall muscle specification in this lineage appears to be different
from that in the other blastomeres. The blastomere C also
produces hypodermis. To test the possibility that the formation
of hypodermis may depend on the MS lineage, which influences the inhibitory activity of the C lineage, I evaluated the
differentiation of the C-derived hypodermis in ablated
embryos. Since the major part of the hypodermis is derived
from the AB lineage and since I am not aware of a reliable
staining method to score the C-derived hypodermis directly, I
analysed the differentiation of hypodermal cells derived from
the C lineage by using the 4-D Microscope. After the ablation
of either EMS or MS, most prospective hypodermal cells did
differentiate into hypodermis. A few hypodermal precursors,
however, underwent additional mitoses (Fig. 4), which could
be taken as an indication that these cells are not specified
normally. The significance of this effect remains to be clarified.
Muscle specification appears autonomous but it is
not
To study the problem of cell determination during embryogenesis, it is essential to know the basic mechanism(s) by which
a certain structure is specified. The interpretation of mutant
phenotypes of developmental genes depends on a detailed
knowledge of the general pathway of cell determination. All
mutant phenotypes of genes affecting descendants of the blastomere P1 have been interpreted in terms of a cell-autonomous
Duelling interactions in Caenorhabditis 2229
specification of the tissues derived from this blastomere
(Kemphues et al., 1985; Schnabel and Schnabel, 1990;
Bowerman et al., 1992a; Mello et al., 1992). The main aim of
this work is to show that the somatic founder cells MS and D,
and possibly C, participate in cell-cell interactions during their
early development.
The three lineages producing body wall muscle can still do
so when the remainder of the embryo is laser ablated. By this
criterion, body wall muscle is specified autonomously (Fig.
8A). If, however, only single blastomeres like ABa or EMS are
ablated, muscle production is suppressed in the MS and D
lineages, respectively. This result clearly contradicts the notion
that body wall muscle is specified autonomously in the
embryonic context. It indicates that in the embryo muscle must
be induced. This contradiction can only be resolved by
assuming that other interactions normally suppress body wall
muscle in the embryonic context. I indeed identified two
lineages producing inhibitory signals. Muscle formation in the
MS lineage is suppressed by the D blastomere. The formation
of body wall muscle in the D lineage itself is suppressed by
the C lineage.
Thus formally four lineages interact during body wall
muscle specification (Fig. 8B). There is strong evidence that
three of the four lineages involved in this network of interactions are affected by the interactions. The question whether the
C lineage is also affected by the interactions remains open.
Analysis of the timing of the interactions showed that the interactions occur at two stages of development. At the 12-cell
stage, the ABa lineage and the MS blastomere interact reciprocally. Both inductions depend on the receptor encoded by the
glp-1 gene (Austin and Kimble, 1987; Priess et al., 1987;
Yochem and Greenwald, 1989; Hutter and Schnabel, 1994;
Schnabel, 1994). This induction specifies features in both
lineages. The MS blastomere induces the left-right asymmetry
within the ABa lineage (Hutter & Schnabel, 1994) and the ABa
lineage induces body wall muscles in the MS lineage
(Schnabel, 1994). This induction, as will be discussed below,
anticipates a counteracting inhibitory signal being part of the
other interactions among the MS, C and D lineage, which occur
about one cleavage later at the late 24- and the early 26-cell
stage. The interaction between the MS and D lineage is
formally also reciprocal. This reciprocity is not created by a
direct cellular contact but in a more complicated way involving
cells lying in between.
These latter interactions occur through or involve the two C
descendants Ca and Cp (Fig. 8C) but are qualitatively very
different. In the case of inhibition of MS-derived muscle, the
data are consistent with the notion that the C descendants act
A
Muscle
C
ABa
Fig. 8. A network of duelling interactions in the early C. elegans
embryo. (A) Upon isolation from the remainder of the embryo, the
MS, C and D lineages produce body wall muscles cellautonomously. Nevertheless body wall muscle specification in the
embryo depends on cell-cell interactions. A formal description of the
interactions modulating muscle specification is depicted in (B).
(C) The interactions among the lineages are reciprocal. The ABa
lineage activates body wall muscle in the MS lineage whereas the
MS lineage specifies the left-right asymmetry of the ABa lineage.
The interactions of the MS and D lineage occur through the C
lineage and are very probably transmitted through cell-cell contacts
among the descendants of these lineages. (D) Spaghetti model of the
duelling interactions. The cell-cell interactions activating body wall
muscle do so by inducing states in the MS and C lineages with
altered competence (squares). At the 12-cell stage, the ABa lineage
alters the state of the MS lineage to one that is no longer susceptible
to the inhibitory signal that it is exposed to one cleavage later. The
MS lineage suppresses the competence of the C lineage to inhibit the
muscle production in the D blastomere at the late 24-/early 26-cell
stage of the embryo. Different shapes indicate different states of
competence of blastomeres. The squares are states that do not
participate any more in the interactions. The fonts indicate different
fates of the blastomeres. (ABa) Ground state, left-right symmetric.
(ABa) Induced, left-right asymmetric. (MS) Ground state, normal
fate, susceptible to inhibition. (MS) Induced state, resistant to
inhibition. (MS) Abnormal fate induced, no body wall muscle
produced. (C) Muscle specification in the C lineage is not influenced
by the interactions. (D) Ground state, normal fate, susceptible to
inhibition. (D) Abnormal fate induced, no body wall muscle
produced. The stippled circles enclose the states that participate in
the interactions. Since embryos from mothers homozygous for one
allele (e2141) of glp-1 are left-right symmetric but have normal body
wall muscles an uninduced ABa lineage can still activate muscle in
the MS lineage (Schnabel, 1994). All states of the MS lineage
prevent the inhibitory function of the C lineage. It is currently not
clear whether an inhibited D blastomere is able to suppress body wall
muscle in the MS lineage.
B
MS
D
Muscle
Muscle
ABa
C
P4
C
MS
D
Muscle
Muscle
Left-right
C
ABa
D
MS
Muscle
Muscle
D
Left-right
ABa
C
C
ABa
MS
MS
D
No muscle
Activation
Inhibition
2230 R. Schnabel
as transmitters of the signal which is derived from the D blastomere. The C lineage on its own does not inhibit muscle production in the MS lineage. In this case, the affected partners of
the interactions are interacting through intercalated blastomeres. It was not possible to block the inhibitory signal completely. A possible explanation for this could be that the signal
is transferred along the surface of the blastomeres, which may
not be affected as severely by the ablations as the cytoplasm
and the nuclei of the irradiated blastomeres.
It appears surprising that the disturbance of cell-cell interactions (Figs 3C3, D7, 4E and 6A4) causes a blastomere to
execute a lineage pattern that in its terminal branches is
partially normal and partially abnormal. It is thus possible to
induce fates partially (see also Hutter and Schnabel, 1994, Fig.
2 for another example). This indicates that the cell determination may be labile throughout the whole lineage specification.
This somehow contradicts the general notion that developmental decisions always lead to the establishment of exactly
one fate.
The situation concerning the modulation of muscle production in the D lineage appears to be different from that in MS.
In this case, the most simple assumption is that the interactions
occur directly between the activator and inhibitor and not
directly with the affected D lineage. The MS lineage suppresses the inhibitory function of the inhibiting C lineage. The
effect is thus indirect. It appears that the C lineage displays a
ground state that inhibits muscle production in the D lineage.
This state is altered by the MS lineage to another state which
is no longer inhibiting the D lineage and D can now act
autonomously (Fig. 8D).
The interacting cells all have direct cell-cell contacts, which
suggests that the signals are mediated by these contacts. This
can be proven by showing that an experimental alteration of
the contacts between blastomeres also alters the induction of
fates, as it was demonstrated for the induction of the ABp fate
by P2 and the induction of left-right asymmetrical fates in the
ABa lineage by MS (Hutter and Schnabel, 1994; 1995;
Moskowitz et al., 1994). This experiment depends, however,
on the existence of other competent, i. e. equivalent blastomeres that are possibly induced by altered contacts. An interesting feature of the cell-cell interactions described here is that
they do not serve to discriminate between equivalent cells,
which is normally the purpose of ‘instructive’ inductions. In
the classical terms, they qualify rather as ‘permissive’ or in the
case of the inhibitory signals as ‘nonpermissive’ inductions
(for review see Slack, 1991). Because in ‘permissive’ inductions equivalent cells do not exist, the appropriate experiments
to test whether the interactions indeed are mediated through
contacts cannot be executed.
A further interesting feature of the interactions is that they
all appear to be independent from each other. They occur at
different embryonic stages and by interfering with one of the
interactions one does not necessarily influence any of the other
interactions. The ablation of ABa suppresses the production of
muscle in the MS lineage but it does not affect the general competence of the MS lineage to interact with the C lineage. The
same is true for the interactions in the posterior of the embryo.
Ablation of C removes the inhibition of D, but D still produces
the signal inhibitory to the MS lineage, since the ablation of C
only partially hinders the signal suppressing muscle in the MS
lineage.
Classically nematodes were considered to develop cellautonomously (for review see zur Strassen, 1959). However,
this notion was already disputed at the beginning of this
century (Boveri, 1910). In recent years, it was well established
that the AB lineage of the C. elegans embryo is specified by
at least five different inductions following the embryonic axes
(Priess and Thomson, 1987; Schnabel, 1991; Wood, 1991;
Bowerman et al., 1992b; Hutter and Schnabel, 1994; Mango et
al., 1994; Mello et al., 1994; Moskowitz et al., 1994; Hutter
and Schnabel, 1995; Hutter and Schnabel, unpublished data).
The P1 lineage, however, has been typically viewed as developing autonomously (Laufer et al., 1980; Cowan and
McIntosh, 1985; Edgar and McGhee, 1986; Kemphues et al.,
1988; Schierenberg, 1988; Schnabel and Schnabel, 1990;
Bowerman et al., 1992a; Mello et al., 1992). This view was
first challenged by Schierenberg (1987) and later by Goldstein
(1992), who suggested that the P2 blastomere interacts with
EMS blastomere to permit the expression of its potential to
express intestine. My previous work (Schnabel, 1994) and this
work now suggest that all P1-derived somatic founder cells
participate in inductions.
Different pathways for body wall muscle
specification
The work presented here shows that body wall muscle specification in the MS and D lineages is susceptible to cell-cell
interactions, though in each case differently, whereas the
muscle specification in C appears to be cell-autonomous. This
indicates that there exist possibly three different pathways for
body wall muscle specification in the early C. elegans embryo.
Genetic evidence for two different pathways of body wall
muscle specification also emerged earlier during the course of
the analysis of the gene skn-1 (Bowerman et al., 1992a, 1993;
Mello et al. 1992). Since this gene is required for the specification of the mother of the MS blastomere, the EMS blastomere, it probably acts upstream of the MS specific pathway
for muscle specification.
How are the functions of blastomeres integrated?
The observations presented here all address the question by
which means the early functions of the blastomeres, for
example the competence to emit and/or to receive signals, are
related to the general identities of blastomeres which are
reflected by the lineage pattern and tissues produced by these
blastomeres. Two extreme scenarios may help to clarify the
problem. One scenario is that all functions of a blastomere are
strictly coupled. The blastomeres have one identity that integrates all the functions at a certain stage. For example, the MS
blastomere has an integral identity that controls the functions
inducing the AB and the C lineage but also activates the
programs governing the control of the complex lineage pattern
producing the tissues corresponding to the lineage. According
to this scenario, it should not be possible to separate the
different functions of the program of a blastomere easily. This
could only be achieved by interfering with downstream
functions for example by genetic means. This notion has been
described earlier for P1-derived blastomeres using the term
blastomere identity (Schnabel and Schnabel, 1990; Mello et al.,
1992).
The other scenario is that blastomeres have roles during the
early development that are not directly related to their function
Duelling interactions in Caenorhabditis 2231
as precursors for tissues formed later in development. In this
case, it should be possible to separate these functions. Considering the results presented here I favour the latter scenario. It
is possible to interfere e.g. with one part of the function of the
MS lineage without affecting others. One can suppress the production of body wall muscle right after the blastomere is born
without affecting the other features of the lineage, for example
the potential to alter the state of the C lineage.
existed that were just evolving from a nonautonomous to an
autonomous strategy or vice versa. An interesting question is
how such a profound change in the basic mechanism may be
achieved under the very strong constraint that a functional
animal must be maintained during this evolutionary process. It
is feasible that different pathways are maintained or partially
maintained until the fidelity of the new pathway is sufficiently
optimised.
Blastomeres may acquire transient states not
reflected in their own specification
The MS lineage is subjected to two duelling interactions,
which occur at different times of embryogenesis. The activating induction from the ABa lineage that sustains the specification of body wall muscle occurs before the interaction suppressing body wall muscle, which has an effect only if the
activation did not occur. The activating signal therefore alters
the state of the MS lineage to render it insensitive to the second
signal. The phenomenon that two different states exist is also
observed in the C lineage (Fig. 8D). The MS lineage switches
the C lineage from a default state that is inhibitory for the D
lineage into another state with no inhibitory capability. The
two states of the C lineage are, in contrast to the MS lineage,
not reflected in at least profound changes of the differentiation
potential of the C lineage itself (Fig. 4). It thus appears that
blastomeres acquire states in the embryonic context that cannot
be diagnosed directly by evaluating the differentiation
potential of the blastomeres in isolation or even in the embryo
proper. The states are only reflected in their effects on other
cells. This insight has implications for the general interpretation of isolation experiments where primordia or combinations
of primordia are analysed in cell culture. The behaviour of
isolated cells may be very different from that of cells within
the embryonic context. The behaviour of the C blastomere may
serve as an example of such a phenomenon. An isolated C blastomere suppresses muscle formation in an opposed D blastomere but in an intact embryo this will never occur. It is quite
conceivable that similar phenomena may occur in systems
more complex than C. elegans.
I thank Lewis Wolpert for suggesting that the observed phenomena
may reflect the ongoing evolution of cell specification in the embryo
and Tom Cole for supplying the electron micrograph, Heinke
Schnabel, Richard Feichtinger and Harald Hutter for helpful discussions and for critical comments on the manuscript, Thierry Bogaert,
Titus Kaletta, Don Moerman and Thomas Wilm for critically reading
the manuscript.
Why do these interactions occur?
An intriguing question is why this network of duelling interactions should exist. I could not find any obvious purpose for
these interactions using the given methods. I cannot exclude,
however, that there exists a purpose that escapes my attention,
possibly because the current concepts about embryogenesis are
insufficient for such a situation. There is the possibility that
these interactions have no essential purpose in the specification of cells at present. The interactions may simply reflect the
evolution of the cell specification machinery. It is possible that
the C. elegans embryo is just evolving from one specification
system involving many cell-cell interactions to another system
involving an autonomous specification or vice versa. It may be
possible to test this by analysing early embryogenesis in other
nematode species. Recently Sommer and Sternberg (1994)
suggested that vulva formation in two nematode species
Mesorhabditis and Teratorhabditis occurs autonomously,
which contrasts the nonautonomous specification of this
structure in C. elegans. This suggests that identical structures
may be specified by completely different strategies within a
single phylum. Therefore, at some time organisms must have
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(Accepted 28 March 1995)
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