Download An analysis of the response to gut induction in the C. elegans embryo

Development 121, 1227-1236 (1995)
Printed in Great Britain © The Company of Biologists Limited 1995
1227
An analysis of the response to gut induction in the C. elegans embryo
Bob Goldstein
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
SUMMARY
Establishment of the gut founder cell (E) in C. elegans
involves an interaction between the P2 and the EMS cell at
the four cell stage. Here I show that the fate of only one
daughter of EMS, the E cell, is affected by this induction.
In the absence of the P2-EMS interaction, both E and its
sister cell, MS, produce pharyngeal muscle cells and body
wall muscle cells, much as MS normally does.
By cell manipulations and inhibitor studies, I show first
that EMS loses the competence to respond before it divides
even once, but P2 presents an inducing signal for at least
three cell cycles. Second, induction on one side of the EMS
cell usually blocks the other side from responding to a
second P2-derived signal. Third, microfilaments and micro-
tubules may be required near the time of the interaction
for subsequent gut differentiation. Lastly, cell manipulations in pie-1 mutant embryos, in which the P2 cell is transformed to an EMS-like fate and produces a gut cell lineage,
revealed that gut fate is segregated to one of P2’s daughters
cell-autonomously. The results contrast with previous
results from similar experiments on the response to other
inductions, and suggest that this induction may generate
cell diversity by a different mechanism.
INTRODUCTION
induction affects only part of a single cell. The EMS cell’s
response to gut induction is then characterised by determining
how long the signal is presented to EMS and how long EMS
is capable of responding, whether both sides of EMS can
respond to inducers at the same time, whether microfilaments
and microtubules are required near this time, and the potential
role of induction in a mutant where a second cell develops like
EMS and produces a gut cell lineage. Possible mechanisms by
which gut induction may generate cell diversity are discussed.
Not long ago the early nematode embryo was regarded as an
archetype of mosaic development, in which cell interactions
played little or no role in early development. Diverse cell types
were believed to be generated almost entirely by segregating
specific cell components to one daughter cell at each division
(see for example, Nigon, 1965; Laufer et al., 1980; for a contrasting view see Guerrier, 1967). This view has since changed,
as several cell-cell interactions have been found to play a
critical role in generating cell diversity in early C. elegans
development, and also because nematode embryos develop in
more diverse ways than the initial evidence had suggested
(Malakhov, 1994).
The induction analysed here is the induction of the gut cell
lineage in C. elegans. Gut specification allows a study of
induction at the level of single cells; the gut (or ‘intestine’)
derives from all of the progeny of a single blastomere of the
eight cell stage, the E cell. During the four cell stage P2 induces
EMS to produce the gut from its E lineage (Fig. 1). This interaction occurs one cell division before the gut founder cell (E)
is generated (Goldstein, 1992). Although P2 normally contacts
the presumptive E side of EMS, the presumptive MS side of
EMS is also capable of becoming the gut founder cell, when
P2 is moved to contact it instead (Goldstein, 1993).
Previous results have suggested that this induction is
unusual, in that it polarises a single cell (EMS), inducing gut
only in the side it contacts (Goldstein, 1993). This led to
interest in the mechanism by which gut induction generates cell
diversity between E and MS. Here differentiation is assayed
from uninduced EMS cells to determine directly if gut
Key words: C. elegans, EMS cell, induction, gut differentiation,
pie-1
MATERIALS AND METHODS
Strains
The experiments employed the wild-type strain N2 Bristol and the
pie-1 strain pie-1(zu154) unc-25(e156)/qC1 [dpy-19(e1259) glp1(q339)]III under standard conditions (Wood, 1988). Embryos from
mothers homozygous for maternal-effect loss of function mutations
in the pie-1 gene are referred to as pie-1 mutant embryos for convenience. skn-1 and mex-1 mutant embryos are referred to similarly.
Cell manipulations, culture, enzyme histochemistry and
antibodies
Manipulations were carried out as described previously (Goldstein,
1992, 1993). Methods for removing the eggshell and vitelline
membrane and for culturing isolated cells are described in Edgar and
Wood (1993). Cells were cultured in Edgar’s Growth Medium (EGM)
for 12-24 hours before assaying for differentiation. Cell lineaging
employed a multiple focal plane time-lapse recording system (Hird
and White, 1993). In experiments where gut differentiation was
assayed by both lineaging and following differentiation, lineages were
traced through the time rhabditin granules were apparent. Where
lineage times alone were followed to discern E and MS lineage
1228 B. Goldstein
Fig. 1. Top: Four and eight cell embryos, ventral view, posterior to
the right. Cell types produced by E and MS lineages are shown. The
P2-EMS interaction is indicated by an arrow. Bottom: Cell lineage
from fertilised egg (P0) through 26 cell stage.
timings, lineages were followed in EMS for a minimum of three
divisions and differentiation was followed alone in separate cases.
Where EMS’s two daughters were separated from each other, this was
done 4-7 minutes after EMS began to form a cleavage furrow (cases
done earlier suggest the cleavage furrow is not yet complete, as the
furrow generally regresses to form one cell with two nuclei).
Times before or after cell divisions cited here were measured
to/from the time a cytokinetic furrow was first visible in the dividing
cell. The time during the cell cycle when a manipulation was carried
out was determined by noting the time of the manipulation and the time
of the ensuing division. The time of the preceding division was also
either recorded (in most cases) or estimated as in Goldstein (1993).
When P 2 and EMS are juxtaposed they stick together immediately
upon contact. Their orientations are randomised, and the cells do not
then rotate or move around each other to return to their original
positions. This has been established first by watching for overt signs
of movement in high magnification time-lapse recordings (Goldstein,
1993) and second by placing cells in contact and then looking at a
marker for cell orientation, the position of the centrosomes in EMS
and P2 in live cells and by anti-tubulin immunofluorescence in fixed
cells (unpublished observations).
Assaying for differentiation by immunofluorescence or enzyme histochemistry required fixing cells in 2% paraformaldehyde after gentle
washing, as devitellinised embryos are easily disrupted. Partial
embryos were washed briefly by mouth pipetting through two changes
of a simplified protein-free EGM, which included only the water, stock
salts, Hepes buffer, galactose, base mix stock and disodium phosphate
from the EGM protocol. Paraformaldehyde was prepared in simplified
EGM by first dissolving paraformaldehyde at 20% in water (at 60°C,
with approx. 50 µl 5 M NaOH per 5 ml) and then diluting 1:10 in the
simplified EGM. Embryos were then washed by mouth pipetting
through two changes of M9 (Wood, 1988) or PBS with 0.5% Tween20 and were placed on 0.1% polylysine-treated slides. The fixed
embryos stick readily to glass after washing with M9 or PBS, and
hence were not allowed to sink onto glass surfaces during washes.
Cell isolates were scored for differentiation of each major body
tissue except neural tissue. Gut differentiation was assayed by three
methods. (1) Presence or absence of birefringent rhabditin granules
was determined by viewing embryos in polarised light as described
previously (Goldstein, 1992, 1993; Edgar and McGhee, 1986). Except
where otherwise stated, this alone was used to assay for gut differentiation. (2) Esterase histochemical staining was performed as
described by Edgar and McGhee (1986), except for fixation, which is
described above. (3) The monoclonal antibody 1CB4 stains the gut,
and also sperm and chemosensory neurons (Okamoto and Thomson,
1985). 1CB4-positive cells were identified as gut cells by their large
size. This was also confirmed by noting whether each isolate
contained rhabditin granules.
Pharyngeal muscle cell differentiation was assayed using the
3NB12 monoclonal antibody (Priess and Thomson, 1987). Body wall
muscle differentiation was assayed using a polyclonal antibody to
paramyosin kindly provided by Hiroaki Kagawa. Hypodermal differentiation was assayed using a polyclonal antibody to LIN-26 protein,
which stains some descendants of isolated AB and P2 cells (unpublished observations); the antibody was kindly provided by Michel
Labouesse. The 5-6 monoclonal antibody, which stains body wall
muscle cells, and the 9.2.1 monoclonal antibody, which stains pharyngeal muscle cells (see Mello et al., 1992), also work on formaldehyde-fixed cultured embryos (unpublished observations). Antibodies
were used by standard methods, with 1-hour incubations at 37°C for
primary and secondary antibodies, and thorough washes in phosphatebuffered saline with 0.5% Tween-20, before and after each step.
Immunofluorescence was observed on an MRC600 laser-scanning
confocal microscope (Biorad). Photographs of confocal sections (in
Fig. 2) show only a thin field of cells, roughly representative of the
proportions of staining in the whole isolate.
Differentiation of gut, pharyngeal muscle, body wall muscle, and
hypodermis could be assayed in each isolate by photographing
rhabditin granules immediately before fixation, then using a fluorescein-conjugated secondary antibody for 3NB12, and a rhodamineconjugated antibody for the paramyosin and LIN-26 antibodies. These
two could be distinguished from each other as the LIN-26 antibody
stains nuclei of hypodermal cells, and the paramyosin antibody stains
cytoplasm of body wall muscle cells.
Colchicine (Sigma) was used at 64 µg/ml for one batch, 100 µg/ml
for another. Cytochalasin B (Sigma) was used at 5 µg/ml. Embryos
were permeabilised by completely removing the eggshell and vitelline
membrane. The concentration of each inhibitor was the lowest dose
that prevented cytokinesis consistently in a dose-response assay; half
the dose used did not arrest cleavage consistently.
RESULTS
E and MS both differentiate as MS-like lineages in
the absence of gut induction
Previous results have shown that gut fate is induced in the E
lineage by the P2-EMS interaction. Gut differentiation does not
occur from EMS cells isolated early in the four cell stage, but
contact with a P2 cell will rescue gut differentiation (Goldstein,
1992, 1993). As only gut differentiation could be assayed in
these experiments, it was not clear whether E is the only cell
The EMS cell’s response to induction 1229
whose fate is affected by the interaction. In order to determine
whether MS is also affected by the interaction, and also to
determine what the uninduced state of the E lineage is, differentiation was assayed from the progeny of isolated EMS cells.
The results are described briefly here; the complete results are
shown in Fig. 2.
EMS cells isolated before gut induction had occurred (1013 minutes before cytokinesis began in EMS) divided to form
two daughters, both of which produced body wall muscle and
pharyngeal muscle, but no gut or hypodermis, suggesting that
both daughters differentiate as MS-like lineages, as MS
normally produces this suite of cell types. These findings
confirm previous suggestions made on the basis of the timing
of cell divisions: in the absence of gut induction, E loses its
distinctive cell lineage timing and takes on an early lineage
timing of an MS-like lineage (Goldstein, 1992, 1993).
EMS loses the competence to respond before it
divides; P2 continues signalling
The time when the P2-EMS interaction normally specifies gut
in E is known. This occurs 5-6 minutes into the four cell stage
(this is 9-10 minutes before EMS cleaves, as the EMS cell
cycle is 15 minutes long); isolating EMS later than this usually
will not prevent gut specification (Goldstein, 1992). If P2 and
EMS are not in contact at this time, placing them in contact
later might still rescue gut differentiation. Here I determine
when contact between the two cells can no longer induce gut
in an uninduced EMS.
EMS and P2 blastomeres were isolated before gut fate had
Fig. 2. Differentiation from the progeny of induced (A-E) and
uninduced (F-I) EMS cells. (A) Gut-specific rhabditin granules.
(B,F) Pharyngeal muscle marker 3NB12 antibody (green) and body
wall muscle marker anti-paramyosin antibody (red). (C,G) Gutspecific esterase histochemical stain (red), only in progeny of the
induced EMS. (D,E,H,I) Differentiation from the progeny of each of
the two daughters of an EMS cell. D and E derive from the two
daughters of an induced EMS cell, (D) from one daughter, (E) from
the other. H and I derive similarly from the two daughters of an
uninduced EMS cell. (D) Gut-specific rhabditin granules. All cells in
this isolate produced rhabditin granules. This isolate produced no
pharyngeal muscle or body wall muscle (not shown).
(E,H,I) Pharyngeal muscle marker 3NB12 antibody (green) and body
wall muscle marker anti-paramyosin antibody (red). These isolates
produced no rhabditin granules (not shown). (J) Table showing that
in the absence of gut induction, both daughters of EMS differentiate
as MS-like lineages. gut is gut differentiation, as assessed by three
assays (gran, rhabditin granules; ab, 1CB4 antibody staining; est, gut
esterase histochemical stain); bwm, body wall muscle; phm,
pharyngeal muscle; hyp, hypodermis (see Materials and Methods);
induced, EMSs isolated after gut was induced were isolated 0-7
minutes before cytokinesis began in each EMS cell; uninduced, those
isolated before gut was induced were isolated 10-13 minutes before
cytokinesis began in each EMS cell. Both parts show differentiation
from isolated EMS cells (labelled EMS), and from EMS’s two
daughter cells separated from each other (labelled ‘E/MS’), to find
which daughter(s) produce the tissue indicated. The daughter that
produced gut cells (when there was one) is shown on the left of each
pair. Each + or − represents 5-16 cases; each case resulted invariably
as indicated, except when EMS was isolated 0-7 minutes before
dividing, in which EMS occasionally (8/40 cases) produced only
bwm and phm and no gut or hyp, suggesting that gut has not yet been
induced in these cases, as this occurs when EMS is isolated before
gut is induced.
been induced, and were kept separate for a time, after which
they were placed back in contact, cultured, and later assayed
for gut differentiation. When P2 and EMS were isolated before
gut fate had been induced and were placed back in contact 8
minutes after EMS cleaved (this would be during the eight cell
stage in an intact embryo), gut fate could no longer be rescued
in EMS. In order to rescue gut fate in an early-isolated EMS,
P2 had to be placed back in contact with EMS 3 minutes before
EMS cleaved or earlier (Fig. 3).
1230 B. Goldstein
To determine whether this time represents the time that EMS
loses the competence to respond to P2’s signal, or the time that
P2 stops signalling, cells from embryos of differing ages were
combined. First, uninduced EMS blastomeres were tested to
see if they can respond after this time. An EMS blastomere was
isolated before gut fate had been induced (10-13 minutes
before EMS cleaved), was left in isolation at least 10 minutes,
and then was placed in contact with a younger P2 blastomere
(from an embryo in the first half of its four cell stage). In this
way EMS blastomeres of varying ages could be tested to see
if they can still respond to a signal. This experiment was
carried out six times; 0/6 produced gut, suggesting that EMS
loses the competence to respond 3 minutes before it cleaves.
Second, P2 blastomeres were tested to see if they can induce
later than this time. Each P2 was isolated, was left until after
EMS had cleaved, and was placed in contact with another EMS
that was isolated from a younger embryo, before gut had been
specified. Some of the P2 cells had cleaved once or twice before
being placed in contact with EMS. Eight cases of this experiment were assembled; 5/8 produced gut cells. In some of these
cases, P2 had cleaved twice before being placed in contact with
the uninduced EMS, and yet gut differentiation still occurred.
The results show that P2 and some or all of its descendants
continue to present a gut-inducing signal for some time after
the four cell stage. EMS loses the ability to respond to gut
induction 3 minutes before it divides.
Induction on one side of EMS interferes with
induction on the other side
Previous results showed that either side of EMS can respond
to gut induction (Goldstein, 1993). Here it is determined
whether a single EMS cell can respond to inductions on both
sides. This was tested in two ways: by presenting a single P2
cell sequentially to two sites on an EMS cell, and by sandwiching an EMS cell between two P2 cells.
Presenting P2 sequentially to two sites on EMS
P2 was moved to a random orientation after gut specification
had initially occurred (Fig. 4A). To do this, EMS cells were
isolated after gut fate had already been induced, 5-9 minutes
before EMS cleaved. P2 was then placed onto EMS. This randomises the orientation of cell contact (see Materials and
methods). After EMS cleaved, whether one or both of EMS’s
daughters produced gut was determined by two methods. In 12
cases, lineage times were recorded from EMS. In 10/12 cases,
only one of EMS’s daughters took on E-like lineage times, and
the other took on MS-like lineage timing. In the other 2/12
cases, neither cell took an E-like lineage timing. In an additional 13 cases, the two daughters of EMS were separated and
cultured independently. In all 13 of these, gut cells differentiated from only one of the daughters. By both assays, when a
gut lineage was established it was always established in only
one daughter of EMS. Despite P2 being presented to two sites
with a timing that should allow two inductions to occur, in no
case did both of EMS’s daughters produce the gut lineage.
Which daughter of EMS gave rise to the gut lineage is of
interest because it can reveal for how long the site of gut specification, once established, is movable. As P2 was moved to a
random site on EMS, in approximately half of the cases P2 will
have been placed on a region that normally would have become
time (minutes)
Fig. 3. P2-EMS contact can rescue gut specification up to 3 minutes
before EMS cleaves. A timeline is shown of the four to eight cell
stage; the time that EMS cleaves is at zero. Each bar represents one
case where P2 and EMS were isolated before gut was specified (more
than 10 minutes before EMS cleaved) and were placed back in
contact at some time later, except the top bar, which shows that
isolating EMS 10 or more minutes before EMS cleaved prevents gut
differentiation (Goldstein, 1992). The bars below show that rescuing
gut differentiation requires placing P2 back onto EMS at least 3
minutes before EMS cleaved. In these cases it was additionally
confirmed that gut differentiation occurred in the daughter of EMS
which derived from the side of EMS secondarily contacting P2
(11/11 cases by following differentiation of rhabditin granules after
isolating EMS’s two daughters, 4/4 by following the lineage timing
of EMS’s two daughters). Additional results described in text show
that EMS loses the ability to respond 3 minutes before dividing. P2
signals throughout its cleavage cycle and passes the ability to signal
to at least some of its descendants for the next two rounds of cell
division. The possibility that only some of P2’s descendants inherit
the ability to signal might explain why only some (5/8 cases) of the
heterochronic combinations using P2’s daughters or granddaughters
were able to induce gut fate in previously uninduced EMS cells.
MS. When the manipulation was carried out at 5 or 6 minutes
before EMS divides, the gut lineage derived from the side P2
was moved to in half of the cases (2/4 cases at 5 minutes, 2/4
at 6 minutes). In contrast, when the manipulation was carried
out earlier the gut lineage nearly always derived from the side
to which P2 was moved (6/7 cases at 7 minutes, 8/8 at 8-9
minutes). These results suggest that although gut is specified
by 9 minutes before EMS divides (Goldstein, 1992, 1993), this
site can be moved by moving the inducer 7-9 minutes before
EMS divides. During this 3-minute period, removing P2 no
longer prevents gut differentiation, however, moving P2 moves
the site of gut specification.
Sandwiching EMS between two P2 cells
To present inducers to both sides of EMS simultaneously, EMS
blastomeres were isolated before gut had been specified and
The EMS cell’s response to induction 1231
each EMS was sandwiched between two P2 cells (Fig. 4B). In
The results show that although both sides of EMS are
ten cases gut differentiation was then assayed in each of EMS’s
competent to respond to the inducing signal (Goldstein, 1993),
two daughters, isolated after EMS divided. Three additional
when presented with signals on both sides, only one side
cases were assembled differently in order to record which side
generally forms a gut lineage.
of EMS was the original presumptive E side: an extra P2 was
placed onto an intact but
devitellinised four cell stage, on
the presumptive MS side of EMS,
in the first 4 minutes of the four
cell stage. EMS was then lineaged
to determine lineage timings and
which daughter(s) produced gut.
Gut differentiation occurred in
10/13 cases. In nine of these the
gut cell lineage derived from only
one of EMS’s two daughters, and
in one case both daughters
produced complete gut cell
lineages. The finding that only
one daughter usually produced a
gut cell lineage was surprising, as
either side of EMS can consistently respond to P2 (Goldstein,
1993), however it appears they
are generally unable to do so at
the same time.
In the cases where gut cells
were produced from one daughter
of EMS, the two sides of EMS
appeared equivalent in their
potential to become the E lineage:
which daughter produced the gut
could not be consistently
predicted by the age of the two
P2s, the order in which they were
placed on EMS, which P2 was
from the same embryo as the
EMS used, nor which side of
EMS would normally have given
rise to the E lineage. One case is
shown in Fig. 4C-K, where a P2
was added to an intact four cell
stage on the presumptive MS side
of EMS, and the cell which
Fig. 4. Induction on one side of EMS generally inhibits the other side of EMS from responding to a
normally would have been E took second signal. (A,B) The diagram shows two experiments designed to test if EMS can respond to
on MS-like lineage timing. The inducers on both sides, sequentially in A and simultaneously in B. EMS cells are shaded. The timeline
cell that normally would have represents the EMS cell cycle (between the two vertical bars on the ends), which lasts 15 minutes.
been MS took on E-like lineage Each time division marked is 5 minutes. Only one daughter generally produced gut cells. (C-K) An
timing and produced the gut.
EMS cell sandwiched between two P2 cells in an otherwise intact embryo. To do this, a P2 cell from
One potential caveat to these another embryo was added to the presumptive MS side of EMS. In the case shown the presumptive MS
experiments is that secondary lineage (labelled ‘MS’) produced the gut cell lineage. Which P2 is the extra one was determined by
interactions between EMS’s noting slight asymmetries in the embryoid immediately after the manipulation, and was confirmed by
daughters might affect results, for watching P2 cell cycle times. (C) Ventral view after EMS divided. (D) Lower focal level at same time,
example by lateral inhibition as showing the four daughters of AB, on which the embryoid is resting. (E) ‘E’ and ‘MS’ after dividing.
This next cell cycle lasted 37 minutes in the two ‘E’ cells, 54-56 minutes in the two ‘MS’ cells; normal
occurs between developing vulval cell cycle times appear switched. (F) The presumptive E lineage after its second division. (G) Lower
cells (Sternberg, 1988). This pos- focal level at the same time, showing two presumptive MS cells. (H) Approximately 12 hours later.
sibility is unlikely, since isolating (I) Same view, under polarised light, showing gut-specific rhabditin granules. (J,K) are as (H,I),
EMS’s daughters from each other showing a different focal plane. The gut cells derived from the entire presumptive MS lineage. Note
after EMS divided did not affect gut cells are larger than other cells; this is also the case in the E lineage of normal embryos, as the E
lineage goes through relatively few cell divisions. Bar, 10 µm.
results (see above).
1232 B. Goldstein
Microfilaments and microtubules
Microfilaments and microtubules appear to play a crucial role
in cytoplasmic localisation in many systems including C.
elegans (see Sardet, 1994 for review). To investigate the role
of the cytoskeleton in the response to gut induction, it was
determined whether microfilaments or microtubules are
required during the four cell stage for gut to differentiate.
Although cell divisions do not occur, cleavage-arrested cells of
a variety of embryos commonly continue to differentiate
(Whittaker, 1973). The role of microfilaments has been tested
previously with varying results (Laufer et al., 1980; Cowan and
McIntosh, 1985; Edgar and McGhee, 1986).
Embryos were devitellinised at various stages, placed in
microfilament- or microtubule-depolymerising agents
(cytochalasin or colchicine, respectively), and were cultured
through the time that gut cells would normally differentiate.
Whereas depolymerising microfilaments in eight to twenty cell
stage embryos had no effect on subsequent gut differentiation,
treating four cell stage embryos nearly always prevented gut
differentiation (Fig. 5A). Embryos treated during the six cell
stage, which represents the last 3 minutes of the EMS cell
cycle, produced gut in about half of the cases. The results
suggest that microfilaments may be required during the four
cell stage for gut differentiation to occur, and that they may no
longer be required soon after this time. Results with microtubule-depolymerising agents were similar (Fig. 5B), suggesting that microtubules also may be required during the four cell
stage.
Gut specification in pie-1 mutant embryos
EMS fate has been proposed to be specified by the activity of
the SKN-1 protein (Bowerman et al., 1992a; 1993). The pie-1
gene product is believed to repress SKN-1 activity in the P2
cell, preventing P2 from developing as an EMS-like cell. In
pie-1 mutant embryos, P2 generally develops as EMS normally
does (Mello et al., 1992). Thus pie-1 mutant embryos produce
two EMS-like lineages, one from EMS and another from the
cell that would normally be P2. Two gut cell lineages result,
one from a daughter of EMS and one from a daughter of P2.
As there is no P2-like blastomere in pie-1 mutant embryos,
it was of interest to examine whether the two gut lineages
produced in these embryos are specified via induction. The
simplest apparent explanation was that an EMS blastomere
might normally possess the ability to induce gut but cannot
induce gut in itself, perhaps because its signal must be
presented on an apposing membrane. If EMS normally
presents a gut-inducing signal, then the formation of two gut
lineages in pie-1 mutant embryos could be explained by both
of the EMS-like blastomeres inducing gut in each other. To test
this hypothesis EMS cells were isolated from wild-type
embryos in the first 4 minutes of the four cell stage, and were
placed back in contact in pairs. In none of five sets of EMS
pairs did gut differentiation occur, suggesting that EMS does
not in fact present a gut-inducing signal.
Blastomeres were then isolated from pie-1 mutant embryos,
to determine directly if EMS and the EMS-like P2 cell require
cell-cell interactions to produce gut cells. EMS blastomeres
isolated in the first 5 minutes of the four cell stage did not
produce gut cells (0/7 cases), whereas P2 blastomeres isolated
at the same time did (7/7 cases). The gut cells occupied roughly
half the volume of each P 2 isolate (Fig. 6). The results demon-
Fig. 5. Microfilaments and microtubules may be required near the
time of the induction for subsequent gut differentiation. Embryos
were treated with cytochalasin (A), colchicine (B), starting from the
stage indicated and continuing until gut differentiation was assayed.
Positions on the Y axis represent the percentage of the cleavagearrested embryos at each stage in which gut differentiation occurred.
The timeline begins at the beginning of the two cell stage. The
transition from the four to six cell stage (when ABa and ABp divide)
occurs near when EMS normally loses the ability to respond (3
minutes before cleaving), and is used to separate embryos placed in
inhibitor in the first 12 minutes of the EMS cell cycle from those
placed in inhibitor in the last 3 minutes of the EMS cell cycle. 97
embryos were used in A; gut differentiation occurred in embryos
treated from the four cell stage in 2/29 cases, and in those treated
from the six cell stage in 11/17 cases. 96 embryos were used in B;
gut differentiation occurred in embryos treated from the four cell
stage in 0/17 cases, and in those treated from the six cell stage in
4/12 cases.
strate that in pie-1 mutant embryos gut is still induced in EMS,
presumably by ‘P2’, however P2 produces gut cells independent of interactions with other cells.
DISCUSSION
This work has characterised some aspects of an unusual
induction. Gut induction generates cell diversity between the
two daughter cells of a single responding blastomere.
Uninduced EMS cells produced two MS-like lineages, suggesting that the induction generates polarity in EMS before it
divides. Heterochronic cell combinations showed that EMS
can only respond to gut induction in a single cell cycle,
whereas P2 can signal over at least three cell cycles. Induction
on one side of EMS inhibited the other side of EMS from
responding to a second P2-derived signal. Microfilaments and
The EMS cell’s response to induction 1233
Fig. 6. pie-1 mutant embryos: gut fate is established cell-autonomously in one daughter of the P2 cell. (A,B) EMS cells from pie-1 mutant
embryos isolated in the last 7 minutes (A) or the first 5 minutes (B) of the EMS cell cycle, which lasts approximately 15 minutes. Isolating
EMS early prevents gut induction. (C,D) P2 cells from pie-1 mutant embryos isolated at the same times as above. Isolating P2 early did not
prevent gut specification in P2. Bar, 10 µm. (E) Model for E and MS specification in wild-type embryos, mutants, and manipulations, from
results presented by Mello et al. (1992), by Bowerman et al. (1992a, 1993), and here. Results from intact embryos are shown at left, from
isolated cells at right. Shading summarises how cells differentiate in each situation. MS-like fate is indicated by light shading, E fate (gut) by
dark shading. Unshaded cells differentiate as other lineages. Note that all of the cells with shading are the ones believed to have active SKN-1
protein (Bowerman, 1993). In wild-type embryos (WT), gut fate is induced in the posterior side of EMS. In pie-1 mutant embryos, both P2 and
EMS have EMS-like fate, although occasionally both of P2’s daughters produce gut lineages (Mello et al., 1992). In mex-1 mutant embryos,
ABa and ABp each have SKN-1 protein and produce pairs of cells with MS-like fates (Mello et al., 1992; Bowerman et al., 1993). Results
presented here suggest that in wild-type embryos and in pie-1 mutants the P2-EMS interaction causes a segregation event in the EMS, and in
pie-1 mutants gut fate is segregated autonomously in P2. In mex-1 mutants, ABa and ABp might not form E-like lineages because they may
lack the ability to segregate gut fate.
microtubules appeared to be required near the time of the
induction. Lastly, in pie-1 mutant embryos, where the P2 cell
has an EMS-like fate, gut fate is segregated to one daughter
cell-autonomously.
Implications of results
Two interactions serve to elaborate the anterior-posterior axis of
the C. elegans embryo: on the ventral side of the embryo, E and
MS develop distinctly because of the P2-EMS interaction. On
the dorsal side, ABa and ABp develop distinctly because of the
P2-ABp interaction (Bowerman et al., 1992b; Mello et al., 1994;
Hutter and Schnabel, 1994; Mango et al., 1994a; Moskowitz et
al., 1994). Hence P2 is the source of two inductions during the
four cell stage. Mango et al. (1994a) have suggested that P2 may
present two distinct signals to EMS and ABp.
Gut induction and the mechanism of generating cell
diversity
Previous work on inductions in many embryos has elucidated
a general mechanism by which many inductions generate cell
diversity: of the cells that are competent to respond to an
induction, only some encounter the inducing molecule(s) (see
Slack, 1991 for review). As a result, these cells differentiate
along a different pathway than the cells that do not encounter
the inducing molecule. In vertebrate embryos, inducing
molecules are commonly growth factors which diffuse many
cell diameters. After some time cells lose their competence to
respond, and only the cells that have encountered the inducer
before this time are affected by the induction. Unlike gut
induction in C. elegans, these inductions commonly involve
large numbers of cells, which can respond through several cell
cycles (Gurdon et al., 1985; Dale et al., 1985; Jones and
Woodland, 1987). In invertebrate embryos the inducer is
commonly presented on cell surfaces, and only the cells
competent to respond that contact the inducing cell(s) are
affected. Other cells are also competent to respond, but do not
contact the inducing cell(s) (see Slack, 1991 for review).
Gut induction is peculiar in that the area which is competent
to respond is a single cell (EMS), and only one side of this cell
is affected by the induction. One way of explaining how gut
induction generates cell diversity is via the type of mechanism
described above, in which only part of EMS encounters an
inducing molecule and develops differently than the other part,
on this basis. Alternatively, the inducer might cause a directed
movement of particular components, in the cytoplasm or the
membrane, to one side of the responding cell, causing one
daughter to differentiate differently than the other (Fig. 7).
Several of the experiments presented here address these
models. The two models are not mutually exclusive, and many
variations on the models are possible; the key point addressed
here is whether or not the induction causes a directed
movement of cell components to one side of the responding
cell, and by doing so generates cell diversity.
Either side of EMS is competent to respond to gut induction
(Goldstein, 1993). If P2 causes the segregation of particular
components to one side in EMS, this might interfere with the
other side’s ability to respond to a second signal. This was
found to be the case, in two types of experiments. First, moving
the P2 cell after gut had been specified moved the site of gut
specification for the first 3 minutes (6-9 minutes before EMS
1234 B. Goldstein
divided), and no longer had an effect if moved during the next
3 minutes (3-6 minutes before EMS divided). Throughout this
6 minute interval, an uninduced EMS can respond to gut
induction, however moving P2 did not establish a second site
of gut specification (Fig. 7A). Second, when EMS was sandwiched between two P2 cells, either of EMS’s daughters could
form the gut founder cell, however only one generally did. The
findings show that as well as inducing gut fate in the side of
EMS it contacts, P 2 has a detectable effect on the other side of
EMS, inhibiting it from responding to a second gut inducing
signal. In other inductions (where many cells can respond to
the inducer) presenting the inducer to more of the area
competent to respond results in more of the induced tissue, in
both vertebrate embryos (Jacobson, 1963) and invertebrate
embryos (for example, van Vactor et al., 1991).
Both microfilaments and microtubules were found to be
required at the four cell stage for subsequent gut differentiation. Depolymerising microfilaments or microtubules later
than this time, during the eight cell stage or later, had little
effect on gut differentiation. Although the timing of the
requirement for microtubules and microfilaments is suggestive,
this result is difficult to interpret. Microfilaments and microtubules, rather than functioning to localise a cell component,
could have a non-motile function, for example to stabilise the
localisation of a cell component. Also, previous experiments
testing the role of microfilaments in gut specification have had
varying results (Laufer et al., 1980; Cowan and McIntosh,
1985; Edgar and McGhee, 1986), most roughly consistent with
those presented here, except for one in which nearly every
embryo produced gut after cytochalasin treatment beginning in
the four cell stage (Cowan and McIntosh, 1985). An interesting finding in some of these studies is that cytochalasin
treatment beginning at the two cell stage prevents gut differentiation less effectively than treatment beginning at the four
cell stage. This might be explained if an inhibitor of gut specification were normally segregated into MS, and it was too
diluted to be effective in the large P1 cell of the two cell stage,
however other explanations are equally plausible.
The role of cell-cell interactions was examined in specifying
gut in pie-1 mutants, where the P2 cell has EMS-like fate and
also produces a gut lineage from one daughter cell. Results
showed that in the EMS-like P2 cell, gut fate is segregated to
one daughter independently of cell contact, yet the normal EMS
cell still requires a cell-cell interaction to establish gut fate in
one daughter. The possibility remains that an isolated pie-1 P2
cell produces a signal on its future P3 side and induces its own
future P3 side to produce gut cells; this appears unlikely, however, since in several experiments no evidence has been found
for a signal produced only on part of P2; P2 appears to present a
signal on all sides (Goldstein 1992, 1993, and unpublished
observations). The result, that in pie-1 mutants gut fate appears
to be segregated cell-autonomously to one daughter of the P2
cell, lends credence to the notion that the induction normally
causes a segregation of cell components in EMS.
The results of experiments thus far are consistent with the
hypothesis that contact with P2 causes a cytoplasmic segregation event in EMS. No cell components however have been
seen to segregate in EMS. Following granule movements in
EMS in normal embryos and in P2-EMS combinations, as has
been done for the segregation event occurring before first
cleavage (Hird and White, 1993), showed no apparent directed
granule movements (unpublished observations). Granules of
the size class found in C. elegans embryos might not however
move in cells as small as EMS (S. Hird, personal communication).
Gut induction might cause the segregation of a gut determinant, distributed throughout EMS below putative threshold
levels, toward the site of contact with P2. Alternatively an
inhibitor of gut fate might be distributed throughout EMS, and
then segregate away from P2. Although the results in pie-1
mutants point to a cytoplasmic segregation event, the results are
also compatible with segregation via capping of membrane
components. Both cytoplasmic segregation and capping occur
in some cells as a response to an external cue (Sardet et al.,
1994; Taylor et al., 1971). The Numb protein in Drosophila is
segregated prior to cell division and acts as a determinant (Rhyu
et al., 1994); whether a cell-cell interaction is required to segregate Numb in a particular direction is not known. It will be
interesting to see whether any genes required for gut specification encode gene products which are segregated in EMS,
toward or away from the site of contact with P2. Mutations in
genes involved in distinguishing the fates of E and MS have
recently been identified (B. Bowerman, C. Mello, J. Priess, and
B. Draper, personal communication); molecular characterisation of these may shed light on the mechanism of gut induction.
Some other inductions in invertebrates may also affect only
a part of a single responding cell. One such example is the
induction of the mesentoblast in equally-cleaving molluscan
embryos. In this case as in C. elegans gut induction, only one
daughter of the cell which receives the induction has its fate
altered by the induction (van den Biggelaar and Guerrier, 1979;
see Davidson, 1986 for review).
Fig. 7. (A) Summary of events during the EMS cell cycle. (B) Model
for generation of cell diversity by gut induction: the P2-EMS
interaction may cause a segregation of cytoplasmic components
(arrows), making the presumptive E side of EMS (dark shading)
different from the presumptive MS side (light shading). The
experiments here do not address whether the proposed segregation
event would concentrate a gut-specifying factor into the presumptive
E side (as depicted) or a gut-inhibiting factor away from the
presumptive E side.
The EMS cell’s response to induction 1235
Gut specification and the role of SKN-1
The skn-1 gene product has been proposed to act as a determinant for EMS fate, as it is required for EMS-like fate, and
can alter the fate of other cells when mislocalised (Bowerman
et al., 1992a, 1993; Mello et al, 1992). The SKN-1 protein is
normally found in the nucleus of EMS, and also in the nucleus
of P2 (Bowerman et al., 1993). SKN-1 has no apparent normal
function in P2; its activity is believed to be repressed in P2 by
the pie-1 gene product, as mutations in pie-1 can confer an
EMS-like fate on the P2 cell (Mello et al., 1992). P2 also
appears to retain some characteristics of a normal P2 cell in
pie-1 mutants: results presented here suggest that P2 retains its
ability to induce gut in EMS. P2 also inherits P granules, as it
does in wild-type embryos (Strome and Wood, 1983; Mello et
al., 1992).
The phenotype of mex-1 mutant embryos (Mello et al., 1992)
is puzzling, as mex-1 mutant embryos have SKN-1 protein in
the nuclei of the other two cells of the four cell stage (ABa and
ABp) but rather than producing EMS-like lineages, these cells
each produce two MS-like lineages. It appears as though SKN1 activity in the EMS or the P2 cell may promote EMS-like
fate, yet in ABa and ABp only pairs of MS-like cells result.
Mello et al. (1992) have indicated that an additional
mechanism may need to operate for a cell with SKN-1 activity
to produce a gut cell lineage. Results presented here suggest
what the additional mechanism may be: EMS may segregate
gut fate to one daughter cell via an interaction with P2, and P2
(in pie-1 mutant embryos) may segregate gut fate
autonomously; ABa and ABp (in mex-1 mutant embryos) may
be unable to segregate gut fate to one daughter cell, and hence
develop like uninduced EMS cells do, producing pairs of MSlike daughters. In support, the ABa and ABp cells probably
lack the ability to segregate cell components, as their daughters
develop differently from each other by virtue of their positions,
as a result of later cell interactions (Wood, 1991; Mello et al.,
1994; Hutter and Schnabel, 1994; Mango et al., 1994b;
Gendreau et al., 1994). ABa and ABp in mex-1 mutant embryos
alternatively might lack the competence to respond to P2’s gut
induction signal. Both of these interpretations (diagrammed in
Fig. 6E) imply that SKN-1 activity, rather than simply specifying EMS fate, specifies the uninduced EMS fate, which
produces pairs of MS-like daughters. Segregation of gut fate
could then occur in EMS via the interaction with P2, and in P2
(in pie-1 mutants) cell-autonomously.
I thank Gary Freeman, in whose laboratory I began these experiments, for suggestions and support. I thank S. Hird, B. Podbilewicz,
P. Lawrence, S. Hoppler, J. Gurdon, J. White, S. Gendreau, J.
Rothman, S. Mango, C. Mello, B. Bowerman and two anonymous
reviewers for helpful comments on the manuscript, H. Kagawa and
M. Labouesse for antibodies, J. McGhee for sharing reagents, and the
Caenorhabditis Genetics Center, which is funded by the NIH National
Center for Research Resources for worms. This work was supported
by an NSF grant to Gary Freeman, an NIH predoctoral training grant
at the University of Texas, an American Cancer Society postdoctoral
fellowship, and a Human Frontiers Science Program postdoctoral fellowship.
REFERENCES
Bowerman, B., Eaton, B. A. and Priess, J. R. (1992a). skn-1, a maternally
expressed gene required to specify the fate of ventral blastomeres in the early
C. elegans embryo. Cell 68, 1061-1075.
Bowerman, B., Tax, F. E., Thomas, J. H. and Priess, J. R. (1992b). Cell
interactions involved in the development of the bilaterally symmetrical
intestinal valve cells during embryogenesis in Caenorhabditis elegans.
Development 116, 1113-1122.
Bowerman, B., Draper, B. W., Mello, C. C. and Priess, J. R. (1993). The
maternal gene skn-1 encodes a protein that is distributed unequally in early C.
elegans embryos. Cell 74, 443-452.
Cowan, A. E. and McIntosh, J. R. (1985). Mapping the distribution of
differentiation potential for intestine, muscle, and hypodermis during early
development in Caenorhabditis elegans. Cell 41, 923-932.
Dale, L., Smith, J. C. and Slack, J. M. W. (1985). Mesoderm induction in
Xenopus laevis: a quantitative study using a cell lineage label and tissuespecific antibodies. J. Embryol. exp. Morph. 89, 289-312.
Davidson, E. H. (1986). Gene Activity in Early Development. 3rd ed., Orlando,
Florida: Academic Press.
Edgar, L. G. and McGhee, J. D. (1986). Embryonic expression of a gutspecific esterase in Caenorhabditis elegans. Dev. Biol. 114, 109-118.
Edgar, L. G. and Wood, W. B. (1993). Nematode embryos In Essential
Developmental Biology; A Practical Approach (ed. C. D. Stern and P. W. H.
Holland) pp. 11-20. Oxford: IRL, Oxford University Press.
Gendreau, S. B., Moskowitz, I. P. G., Terns, R. M. and Rothman, J. H.
(1994). The potential to differentiate epidermis is unequally distributed in the
AB lineage during early embryonic development in C. elegans. Dev. Biol. (in
press).
Goldstein, B. (1992). Induction of gut in Caenorhabditis elegans embryos.
Nature 357, 255-257.
Goldstein, B. (1993). Establishment of gut fate in the E lineage of C. elegans:
the roles of lineage-dependent mechanisms and cell interactions.
Development 118, 1267-1277.
Guerrier, P. (1967). Les facteurs de polarisation dans les premiers stades du
développement chez Parascaris equorum. J. Embryol. exp. Morph. 18, 121142.
Gurdon, J. B., Fairman, S., Mohun, T. J. and Brennan, S. (1985). Activation
of muscle-specific actin genes in Xenopus development by an induction
between animal and vegetal cells of a blastula. Cell 41, 913-922.
Hird, S. N. and White, J. G. (1993). Cortical and cytoplasmic flow polarity in
early embryonic cells of Caenorhabditis elegans. J. Cell. Biol. 121, 13431355.
Hutter, H. and Schnabel, R. (1994). glp-1 and inductions establishing
embryonic axes in C. elegans. Development 120, 2051-2064.
Jacobson, A. G. (1963). The determination and positioning of the nose, lens,
and ear. III. Effects of reversing the antero-posterior axis of epidermis, neural
plate and neural fold. J. Exp. Zool., 154, 293-304.
Jones, E. A. and Woodland, H. R. (1987). The development of animal cap
cells in Xenopus, a measure of the start of animal cap competence to form
mesoderm. Development 101, 557-563.
Laufer, J. S., Bazzicalupo, P. and Wood, W. B. (1980). Segregation of
developmental potential in early embryos of Caenorhabditis elegans. Cell
19, 569-577.
Malakhov, V. V. (1994). Nematodes: Structure, Development, Classification,
and Phylogeny. Washington and London: Smithsonian Institution
Press.
Mango, S. E., Thorpe, C. J., Martin, P. R., Chamberlain, S. H. and
Bowerman, B. (1994a). Two maternal genes, apx-1 and pie-1, are required
to distinguish the fates of equivalent blastomeres in the early C. elegans
embryo. Development 120, 2305-2315.
Mango, S. E., Lambie, E. J. and Kimble, J. (1994b). The pha-4 gene is
required to generate the pharyngeal primordium of Caenorhabditis elegans.
Development 120, 3019-3031.
Mello, C. C., Draper, B. W., Krause, M., Weintraub, H. and Priess, J. R.
(1992). The pie-1 and mex-1 genes and maternal control of blastomere
identity in early C. elegans embryos. Cell 70, 163-176.
Mello, C. C., Draper, B. W. and Priess, J. R. (1994). The maternal genes apx1 and glp-1 and establishment of dorsal-ventral polarity in the early C.
elegans embryo. Cell 77, 95-106.
Moskowitz, I. P. G., Gendreau, S. B. and Rothman, J. H. (1994).
Combinatorial specification of blastomere identity by glp-1-dependent
cellular interactions in the nematode Caenorhabditis elegans. Development,
120, 3325-3338.
Nigon, V. (1965). Développement et reproduction des nématodes. In Traité de
Zoologie 4, 218-372. Paris: Masson.
Okamoto, H. and Thomson, J. N. (1985). Monoclonal antibodies which
1236 B. Goldstein
distinguish certain classes of neuronal and supporting cells in the nervous
tissue of the nematode Caenorhabditis elegans. J. Neurosci. 5, 643-653.
Priess, J. R. and Thomson, J. N. (1987). Cellular interactions in early C.
elegans embryos. Cell 48, 241-250.
Rhyu, M. S., Jan, L. Y. and Jan, Y. N. (1994). Asymmetric distribution of
numb protein during division of the sensory organ precursor cell confers
distinct fates to daughter cells. Cell 76, 477-492.
Sardet, C., McDougall, A. and Houliston, E. (1994). Cytoplasmic domains in
eggs. Trends Cell Biol. 4, 166-172.
Slack, J. M. W. (1991). From Egg to Embryo. (2nd ed.) Cambridge:
Cambridge University Press.
Sternberg, P. W. (1988). Lateral inhibition during vulval induction in
Caenorhabditis elegans. Nature 335, 551-554.
Strome, S. and Wood, W.B. (1983). Generation of asymmetry and segregation
of germ-line granules in early Caenorhabditis elegans embryos. Cell 35, 1525.
Taylor, R. B., Duffus, P. H., Raff, M. C. and de Petris, S. (1971).
Redistribution and pinocytosis of lymphocyte surface immunoglobulin
molecules induced by anti-immunoglobulin antibody. Nature New Biol. 233,
225-229.
van den Biggelaar, J. A. M., and Guerrier, P. (1979). Dorsoventral polarity
and mesentoblast determination as concomitant results of cellular
interactions in the mollusc Patella vulgata. Dev. Biol. 68, 462-471.
van Vactor, D. L., Cagan, R. L., Krämer, H. and Zipursky, S. L. (1991).
Induction in the developing compound eye of Drosophila: Multiple
mechanisms restrict R7 induction to a single retinal precursor. Cell 67, 11451155.
Whittaker, J. R. (1973). Segregation during ascidian embryogenesis of egg
cytoplasmic information for tissue-specific enzyme development. Proc.
Natl. Acad. Sci. USA 70, 2096-2100.
Wood, W.B. (1988). The nematode Caenorhabditis elegans. Cold Spring
Harbor, New York: Cold Spring Harbor Laboratory Press.
Wood, W. B. (1991). Evidence from reversal of handedness in C. elegans
embryos for early cell interactions determining cell fates. Nature 349, 536-538.
(Accepted 1 December 1994)