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387
Development 129, 387-397 (2002)
Printed in Great Britain © The Company of Biologists Limited 2002
DEV8905
Cell polarity and gastrulation in C. elegans
Jeremy Nance1,2 and James R. Priess1,2,*
1Division of Basic Sciences, Fred Hutchinson Cancer Research Center,
2Howard Hughes Medical Institute, Seattle, Washington 98109, USA
Seattle, Washington 98109, USA
*Author for correspondence (e-mail: [email protected])
Accepted 17 October 2001
SUMMARY
Gastrulation in C. elegans embryos involves formation of a
blastocoel and the ingression of surface cells into the
blastocoel. Mutations in the par-3 gene cause abnormal
separations between embryonic cells, suggesting that the
PAR-3 protein has a role in blastocoel formation. In normal
development, PAR proteins localize to either the apical or
basal surfaces of cells prior to blastocoel formation; we
demonstrate that this localization is determined by cell
contacts. Cells that ingress into the blastocoel undergo an
apical flattening associated with an apical concentration of
non-muscle myosin. We provide evidence that ingression
times are determined by genes that control cell fate, though
interactions with neighboring cells can prevent ingression.
INTRODUCTION
1992; Lin et al., 1995; Rocheleau et al., 1997). These
endodermal and mesodermal precursors must then move from
the outer surface of the embryo into the interior for the
formation of a functional intestine and muscular system.
Animal embryos use several strategies to position
endodermal and mesodermal precursors into their interior, a
process called gastrulation. In many embryos, such as those of
sea urchins and amphibians, the early embryonic cleavages
generate a cluster of cells (the blastula), and a central cavity
(the blastocoel) develops within this cluster. Certain cells
detach from their neighbors on the cell surface and enter the
blastocoel (ingression), or large groups of cells fold into the
blastocoel (invagination and involution) (Gilbert and Raunio,
1997). In some animals, gastrulation occurs without formation
of a blastocoel. In ctenophores, for example, ectodermal cells
spread over and internalize endodermal cells (epiboly) (Komai,
1968; Martindale and Henry, 1999).
Gastrulation in C. elegans begins at the 26-cell stage, when
two endodermal precursors ingress from the surface of the
embryo into the interior. Shortly thereafter, mesodermal
precursors and germline precursors follow the endodermal
precursors into the interior of the embryo (Sulston et al., 1983).
Very little is known about the cellular or molecular basis for
these events. Several mutations have been identified that
prevent or delay ingression of the endodermal precursors, most
of which cause the endodermal precursors to divide
prematurely (Denich et al., 1984; Knight and Wood, 1998).
Inhibiting embryonic transcription also causes the endodermal
precursors to divide precociously and prevents ingression
(Powell-Coffman et al., 1996). Ingression of the endodermal
precursors is likely to require embryonic transcription of one
or more genes in a chromosomal region called the endoderm
determining region, or EDR; a chromosomal deficiency that
Numerous studies on C. elegans embryos have revealed the
basic strategies that establish the anterior-posterior and dorsalventral axes. Axis specification in C. elegans embryos begins
with fertilization of the egg, where the point of sperm entry
defines the posterior pole (Goldstein and Hird, 1996).
Fertilization induces the association of a group of proteins,
collectively called PAR proteins, to either the anterior or
posterior cortex. For example, PAR-3 and PAR-6 associate
with the anterior pole while PAR-2 and PAR-1 associate with
the posterior pole (Boyd et al., 1996; Etemad-Moghadam et al.,
1995; Guo and Kemphues, 1995; Hung and Kemphues, 1999).
The localization of the PAR proteins is interdependent;
mutations in par-3 cause the anterior mislocalization of PAR2, and mutations in par-2 cause the posterior mislocalization
of PAR-3 (Boyd et al., 1996; Etemad-Moghadam et al., 1995).
The dorsal-ventral axis is determined as the embryo divides
from two cells to four (reviewed by Schnabel and Priess, 1997).
The division of the anterior cell generates two initially
equivalent daughters that express a receptor related to the
Notch protein. The division of the posterior cell generates nonequivalent daughters, one of which expresses a ligand for the
receptor. In an apparently random manner, only one of the
receptor-expressing cells contacts the ligand-expressing cell;
this interaction leads to the specification of dorsal cell types
such as hypodermis (skin).
In addition to anterior-posterior and dorsal-ventral axes, the
early embryo must establish an outer-inner polarity. Cells on
the ventral surface of the embryo become committed to
endodermal and mesodermal fates through a combination of
cell signaling events and asymmetrically localized factors
(Bowerman et al., 1993; Bowerman et al., 1992; Goldstein,
Key words: Blastocoel, Apical-basal polarity, Gastrulation,
Ingression, Morphogenesis, Cell fate, Cell shape, Non-muscle
myosin, LIT-1, HMR-1, NMY-2, PAR-2, PAR-3, PAR-6, C. elegans
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J. Nance and J. R. Priess
deletes the EDR delays or prevents ingression (Zhu et al.,
1997). There are at least two genes in the EDR that are involved
in specification of the endodermal cell fate, suggesting a
possible link between cell fate and ingression (Zhu et al.,
1997).
In this report we investigate the cellular basis for gastrulation
in C. elegans. PAR proteins that show anterior-posterior
asymmetry in 1-cell and 2-cell embryos subsequently develop
outer-inner, or apical-basal, polarity as the blastula forms. We
demonstrate that the apical-basal localization of the PAR
proteins is dependent on cell-cell contacts by generating double
embryos with abnormal patterns of cell contact. We show that
ingression is associated with changes in the shape of the
ingressing cells and a redistribution of non-muscle myosin.
Finally, we present evidence that cell fate, rather than cell
position, is the predominant factor regulating ingression,
although steric interactions can play an important role.
MATERIALS AND METHODS
Nematode culture and strains
Nematodes were cultured and manipulated as described (Brenner,
1974). Unless otherwise indicated, experiments were performed on
the wild-type N2 (var. Bristol). The following mutants were utilized:
chromosome II LG II: unc-4(e120) (Brenner, 1974), mex-1(zu120)
(Mello et al., 1992); unc-32(e189) (Brenner, 1974), lit-1(t1512ts)
(Kaletta et al., 1997), unc-45(e286ts) (Brenner, 1974), par-2(lw32)
(Cheng et al., 1995), par-3(it71) (Cheng et al., 1995), lon-1(e185)
(Brenner, 1974); LGV: zuDf2 (Zhu et al., 1997). The following
integrated transgenes containing green fluorescent protein (GFP)
reporters were used: zuIs3 (end-1::GFP) (J. Nance, unpublished),
ruIs32 (pie-1::GFP::HIS-11) (Pratis et al., 2001), pxIs6 (pha4::GFP::HIS-11) (Portereiko and Mango, 2001), itIs153 (pie-1::PAR2::GFP) (Wallenfang and Seydoux, 2000). Mex-1 embryos were mex1 unc-4. Par-2 embryos were par-2 unc-45. Par-3 embryos were par-3
lon-1; zuIs3.
dsRNA-mediated interference (RNAi)
Standard techniques were used to synthesize double-stranded RNA
(dsRNA) from T7 promoter-tagged, PCR-amplified cDNA or
genomic DNA. For analysis of ama-1(RNAi) embryos (2-4 µg/µl
dsRNA made from cDNA region 2876-3450), young adult
hermaphrodites carrying both a maternal (pie-1::GFP::HIS-11) and
zygotic (end-1::GFP) GFP reporter were injected in the gonad or
soaked overnight with dsRNA (Fire et al., 1998; Tabara et al., 1998).
After allowing worms to recover for 30 hours, embryos were analyzed
for end-1::GFP expression. In a representative experiment, the end1::GFP reporter was detectable in only 5/224 ama-1(RNAi) embryos.
For analysis of hmr-1(RNAi) embryos (2.5 µg/µl dsRNA made from
genomic DNA region 13344-13989), approximately 20 hours after
injection of dsRNA into wild-type adults, embryos were collected and
fixed for immunofluorescence. Embryos were immunostained to
confirm HMR-1 depletion.
Embryo handling and analysis
To examine blastocoel formation, 1-cell or 2-cell embryos were
pipetted onto a poly-L-lysine (Sigma)-coated coverslip containing
clay spacers; the coverslip was inverted onto a slide for observation.
In all other experiments 2-cell or 4-cell embryos were mounted for
videomicroscopy on 4% agarose (see Sulston et al., 1983). Isolated
AB blastomeres were mounted in embryonic culture medium (Shelton
and Bowerman, 1996) on slides and covered with coverslips coated
with 1.5% agarose; techniques used to isolate and culture embryos or
individual blastomeres were essentially as described (Shelton and
Bowerman, 1996). Embryo combination experiments were performed
at 15°C. To inhibit transcription, embryonic culture medium was
supplemented with 50 µg/ml α-amanitin (Sigma). Laser ablations
were performed as described elsewhere (Mello et al., 1992).
4D images were acquired as described (Thomas et al., 1996) using
4D Grabber v1.32 software (C. Thomas, Integrated Microscopy
Resource, University of Wisconsin-Madison, USA). Movies were
analyzed using 4D Viewer v4.11 (C. Thomas, Integrated
Microscopy Resource, University of Wisconsin-Madison, USA)
and Nematode Navigator software (kindly provided by J. Pitt,
http://www.fhcrc.org/labs/priess/nn.html). The volume of the
blastocoel and embryo (± s.d.) were calculated using NIH Image1.62
(Wayne Rasband, National Institutes of Health, USA).
Ingressions were scored when a cell sank permanently from the
surface of the embryo. Comparisons of ingression times were based
on the analysis of 4D videorecordings of three embryos; all cells that
ingressed during gastrulation were followed in each embryo.
Ingression times were normalized to cell division timings presented
by Sulston et al. (Sulston et al., 1983) using the interval between the
MSa and MSaaa divisions as a measure of developmental time.
Descriptions of wild-type gastrulation were based on observations of
these and additional 4D videorecordings. Ectopic ingressions in mex1 mutants were scored in the ABpr lineage. lit-1 temperature shifts
were from 15°C to 25°C at the 24-cell stage. Because of the variable
expressivity of the lit-1 mutant phenotype, analysis of MS ingression
was restricted to the 7/11 embryos showing simultaneous ingression
of all wishbone cells. In the E ablation experiments, analysis was
restricted to the anterior MS central cells that do not contact the E
corpse.
Electron microscopy
Mixed-stage embryos were fixed, embedded, sectioned and stained for
transmission electron microscopy (TEM) as described (Priess and
Hirsh, 1986). Several thick sections were cut from each block to
determine the precise stage and orientation of specific embryos.
Sections were analyzed from >50 embryos embedded in three
different blocks. Embryos for scanning electron microscopy were
fixed as for TEM, omitting the tannic acid treatment. Embryos were
transferred by mouth pipet to a poly-L-lysine coated coverslip, then
dehydrated and dried as described (Braet et al., 1997).
Immunostaining and fluorescence microscopy
Embryos were fixed and processed for overnight immunostaining at
4°C as described (Leung et al., 1999). Cultured blastomeres were
fixed in –20°C methanol for 15 minutes, then immunostained at 37°C
for 1 hour. A strain containing the itIs153 reporter was used for
analysis of PAR-2 in cultured blastomeres. Unless indicated
otherwise, immunofluorescence studies were based on the observation
of 15-100 embryos at the indicated stage. The following dilutions of
antibodies/antisera were used: 1:200 mouse anti-GFP (‘α’-GFP;
Clontech); 1:10 rabbit α-HMR-1 (Costa et al., 1998); 1:50 rabbit αNMY-2 (Guo and Kemphues, 1996); 1:10 rabbit α-PAR-2 (Boyd et
al., 1996); 1:10 chicken α-PAR-3 (Tabuse et al., 1998); 1:15 rabbit αPAR-3 (Etemad-Moghadam et al., 1995); 1:10 rabbit α-PAR-6 (Hung
and Kemphues, 1999); 1:1000 rabbit α-PGL-1 (Kawasaki et al.,
1998). Secondary antibodies were conjugated with Alexa Fluor 488
(Molecular Probes) or Cy-3 (Jackson ImmunoResearch Laboratories,
Inc.). In some experiments, DNA was stained with 60 ng/ml 4,6diamidino-2-phenylindole (DAPI).
Confocal images were acquired on a Leica TCS SP spectral
confocal microscope. All other images were acquired using a standard
epifluorescence microscope equipped with a digital camera; some
epifluorescence images were numerically deconvolved using
DeltaVision algorithms (Applied Precision, Inc.).
Analysis of NMY-2 localization
For the analysis of NMY-2 localization in MS descendants, embryos
C. elegans polarity and gastrulation
expressing pha-4::GFP::HIS-11 were stained with antibodies or
antisera against PGL-1, GFP and NMY-2; GFP and PGL-1 staining
were used as reference points to identify specific MS descendants
after fixation. NMY-2 was scored as ‘enriched’ when the apical
concentration in an ingressing cell appeared greater than in other
surface cells, exclusive of dividing cells.
RESULTS
Formation of the blastocoel
Gastrulation in C. elegans involves the movement of a subset
of cells from the ventral surface of the embryo into the
blastocoel. These movements, described here as cell
ingressions, occur over a period of about 3 hours and begin at
the 26-cell stage of embryogenesis. Some, or all, of the
descendants of each of the early embryonic blastomeres ingress
(see Fig. 4A,B). The ingressing cells form tissues such as the
endoderm (descendants of the E blastomere), germline (P4
descendants) and mesoderm (D descendants and a subset of
AB, MS, and C descendants; see Fig. 4A).
Prior to gastrulation, the embryo consists of a hull of cells
one cell in thickness. These cells surround a small, central
cavity called the blastocoel (arrowheads, Fig. 1A). For
convenience, we distinguish three types of membrane surfaces
for each cell. The apical membrane faces the perimeter of the
embryo, the basal membrane faces the blastocoel, and the
lateral membranes face adjacent cells within the hull (Fig. 1A).
In three-dimensional timelapse (‘4D’) videorecordings of
living embryos, the blastocoel forms as the basal surfaces of
diametrically opposed cells gradually detach from each other.
Small openings between basal surfaces are first visible at the
4-cell stage (arrowhead, Fig. 1B) and numerous openings of
variable size are present by the 26-cell stage (arrowheads, Fig.
1C). The blastocoel achieves its maximum volume by the 26cell stage when it measures 450±100 µm3 (n=5). This volume
is only 2% of the volume of the embryo (25,800±980 µm3),
and less than half the volume of a single cell (about 1000 µm3).
Because of the small size of the blastocoel, the basal surfaces
of diametrically opposed cells transiently come into contact
Fig. 1. Blastocoel formation. All images represent sections through
the center of embryos. (A) 26-cell embryo indicating the apical (a),
lateral (l) and basal (b) surfaces of a cell lining the blastocoel
(arrowheads). The E daughters are indicated by asterisks. (B) 4-cell
embryo showing a small basal separation (arrowhead); cell names are
listed. (C) Electron micrograph of a 28-cell embryo where the E
daughters (dashed outline) have partially ingressed. Prominent
spaces between cells are indicated by arrowheads. (D,E) Membrane
contacts at the same, high magnification showing examples of (D) a
small space (arrow) between lateral surfaces and (E) a large space
associated with basal surfaces; arrows in E illustrate close apposition
of membranes around the space. (F) 16-cell partial embryo derived
from an isolated AB blastomere; note the large central cavity
(arrowhead). (G) 16-cell par-3 mutant embryo showing abnormally
large separations (arrowheads) between lateral membranes.
(H) Double embryo after fixation showing a cavity (arrowhead) in
the center of AB descendants; a subset of the eight AB descendants
present are visible and labeled 1 or 2 according to origin. The P1
descendants (MS, E, P3, and C) from each half of the double embryo
are labeled. N, nucleus. Intact embryos (A-C,G) are 50 µm. Bar (for
D,E),1 µm.
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after they separate, temporarily obscuring regions of the
blastocoel. In contrast to the often large spaces separating basal
surfaces (Fig. 1E), lateral surfaces either are juxtaposed or are
separated by only small spaces (Fig. 1D).
How might the various surfaces of embryonic cells become
specified, such that the blastocoel forms at the basal surface?
In the 4-cell embryo, the basal surface corresponds to the
region where the two daughters of the AB blastomere (ABa
and ABp) contact the two daughters of the P1 blastomere
(EMS and P2) (Fig. 1B). We asked whether blastocoel
formation required interactions between specific AB and P1
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J. Nance and J. R. Priess
Fig. 2. PAR localization. (A,B) PAR-2 in 4-cell (A) and 7-cell (B)
embryos. (C,D) PAR-3 in early 4-cell (C) and 8-cell (D) embryos.
(E) 28-cell embryo showing PAR-2 (green), PAR-3 (red) and DNA
(blue); the ingressing E daughters are indicated with yellow asterisks.
The embryo is oriented as in Fig. 4D. (F) Nomarski micrograph of
50-cell embryo indicating cell surfaces facing the blastocoel
(arrowheads). (F′) PAR-3 localization in embryo shown in F. PAR-3
is localized to surfaces facing the blastocoel (arrowheads) in addition
to localization at the apical surface (arrow). (G) PAR-3 in an 8-cell
ama-1(RNAi) embryo. (H) PAR-2 in an 8-cell par-3(it71) embryo;
PAR-2 is localized to all cell surfaces (arrows). In A-E and G the
germline precursor is indicated with a cyan asterisk.
descendants by separating AB from P1 at the 2-cell stage and
allowing each blastomere to develop in isolation (n=8). The
AB descendants generated by the first two cycles of cell
division formed a tightly adherent spherical cluster of cells. By
the third cycle of cell division, the clusters developed a central
cavity of variable size that resembled the normal blastocoel
(Fig. 1F). Because in these experiments P1 descendants form
a linear array of cells rather than a spherical cluster (see Fig.
1H) (Schierenberg, 1987), we did not examine the behaviour
of these cells further. We asked whether AB descendants from
separate embryos could generate a blastocoel by combining
two 2-cell embryos, or two early 4-cell embryos, head to head
with the anteriormost surfaces of the AB cells in contact
(n=16). At the point of contact, the surfaces of the combined
blastomeres flattened and adhered tightly. All of these ‘double’
embryos developed a central cavity of variable size surrounded
by both sources of AB descendants (labeled 1 and 2 in Fig.
Fig. 3. PAR localization in double embryos. Double embryos were
immunostained for the PAR protein listed above each panel. The
subset of AB descendants visible are indicated by 1 or 2, according
to origin, and names of some of the P1 descendants are indicated.
Both germline blastomeres (P2) are indicated with asterisks in A.
(C,D) Upper and lower focal planes of the same double embryo.
Note the blastocoel visible at the center of the 1 and 2 cells in C. At
the time of fixation, double embryos were equivalent to the following
single embryo stages: (A) 4-cell plus 4-cell, (B) 8-cell plus 8-cell,
(C,D) 7-cell plus 8-cell. Bar, 10 µm.
1H). We draw several conclusions from these experiments.
First, the surfaces of cells at the 2-cell and 4-cell stages of
embryogenesis are adhesive; thus this adhesion must be
overcome for cell surfaces to separate during blastocoel
formation. Second, interactions between AB descendants are
sufficient to form a blastocoel. Finally, we conclude that cell
contact can reorient the polarity of AB descendants; in double
embryos a blastocoel can form at a surface that would normally
have been an apical surface.
PAR proteins and apical-basal polarity in early
embryos
Analysis of the PAR proteins has focused primarily on their
anterior-posterior localization at the 1-cell stage and their roles
in subsequent anterior-posterior polarity (see Introduction).
After cell division begins, however, this anterior-posterior
asymmetry is reiterated only in the lineage of cells that form
the germline (Boyd et al., 1996; Etemad-Moghadam et al.,
1995; Guo and Kemphues, 1995; Hung and Kemphues, 1999)
(the germline cells P2 and P3 are indicated by cyan asterisks
in Fig. 2). In contrast, some somatic (non-germline) cells show
an apical-basal polarity in PAR localization (Boyd et al., 1996;
Etemad-Moghadam et al., 1995; Guo and Kemphues, 1995;
Hung and Kemphues, 1999). By the 4-cell stage, the posterior
PAR protein PAR-2 is localized to basolateral surfaces (arrows,
C. elegans polarity and gastrulation
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Fig. 2A,B), and is not detected on the apical surfaces of
somatic cells. By the middle of the 4-cell stage, the anterior
PAR proteins PAR-3 and PAR-6 are present over the entire
cortex of each somatic blastomere (arrows, Fig. 2C). However
by the end of the 4-cell stage and at later stages PAR-3 and
PAR-6 are concentrated in a broad ‘cap’ centered on the apical
surface (arrow, Fig. 2D). The apical-basal polarity of the PAR
proteins persists through early gastrulation, although the level
of PAR-2 diminishes (Fig. 2E). After gastrulation begins, cells
in the interior of the embryo accumulate PAR-3 and PAR-6 on
their blastocoel-facing surfaces (arrowheads, Fig. 2F,F′); these
and later changes in PAR distribution were not analyzed further
(see also Leung et al., 1999).
We used 4D videomicroscopy to examine formation of the
blastocoel in par-2 and par-3 mutant embryos. par-2 mutant
embryos (n=6) developed a central cavity resembling the
normal blastocoel (data not shown). par-3 mutant embryos
(n=6) also developed a central cavity, but in addition showed
frequent separations between the lateral membranes of cells
(arrowheads, Fig. 1G); these lateral separations persisted
through several cell divisions. The lateral separations in par-3
mutant embryos were comparable to the spaces observed
between the basal surfaces of cells in wild-type embryos, but
were much larger than the spaces normally present between
lateral surfaces. These results suggest that PAR-3 is required,
directly or indirectly, for the normal adhesiveness of lateral
membranes.
Apical-basal polarity of PAR proteins requires cell
interactions
How do the PAR proteins switch from anterior-posterior
polarity to apical-basal polarity? Germline blastomeres, which
maintain anterior-posterior PAR polarity during the early
embryonic stages, are transcriptionally quiescent, while
somatic blastomeres that switch to apical-basal asymmetry are
transcriptionally active (Seydoux et al., 1996). However, we
found that PAR-3 and PAR-2 switch from anterior-posterior to
apical-basal polarity after embryonic transcription is prevented
by double-stranded RNA inhibition (RNAi) of the ama-1 gene
[Fig. 2G and data not shown; the ama-1 gene encodes the large
subunit of RNA polymerase II (Rogalski and Riddle, 1988)].
In addition, a blastocoel appeared to form normally in ama1(RNAi) embryos (n=6) and in wild-type embryos exposed to
α-amanitin (n=4). Thus apical-basal PAR polarity, as well as
blastocoel formation, appears to be determined by maternally
provided gene products.
We asked whether cell contacts determine apical-basal PAR
polarity by constructing and analyzing double embryos with
ectopic cell contacts, as described above. 2-cell embryos were
joined head to head, then allowed to develop between 15 and
45 minutes (1-3 additional cell cycles) before fixation. We
found that PAR-2 was excluded from the apical regions of
somatic blastomeres in the double embryos, but was localized
to all basal and lateral membranes including ectopic sites of
cell contact (n=6; arrow, Fig. 3A). In contrast, PAR-3 and PAR6 were localized predominately to the contact-free, apical
surfaces of somatic blastomeres (n=6 and 8, respectively; Fig.
3B-D). As in wild-type embryos, there appeared to be a cap of
PAR-3 and PAR-6 toward the center of the apical cortex.
We asked if PAR localization was dependent on HMR-1, a
C. elegans homologue of vertebrate cadherin; HMR-1 is
Fig. 4. Cell ingression. (A) Schematic lineage diagram of early
blastomeres indicating AB and P1 descendants. (B) Lateral view of a
three-dimensional model of nuclei at the 26-cell stage; anterior is
left. The subset of descendants of the early blastomeres that ingress,
or give rise to ingressing cells, are indicated by the color scheme in
A; non-ingressing cells (a subset of AB descendants) are shown in
grey. (C) Two diagrams of the ventral surfaces of embryos outlining
the regions where various cells ingress and listing the periods of cell
ingressions in minutes from the 2-cell stage. The left diagram
indicates ingression of the E daughters and the MS ‘wishbone’
descendants (red), and the right diagram indicates later ingressions
including the MS ‘central’ descendants (red oval). (D-G) Nomarski
light micrographs of representative stages of gastrulation; times as
indicated. (D) 28-cell embryo; lateral view as in B. An MS
descendant (red arrowhead; MSap) and P4 (cyan arrowhead) are
shown spreading across the apical surfaces of the E daughters
(yellow asterisks). (E) Ventral view showing MS ‘central’
descendants (red asterisks) and a subset of C descendants (green
asterisks) prior to ingression. The arrow indicates the cleft created by
ingression of the D descendants. (F) Ventral view showing part of the
cleft created by ingression of the MS descendants; neighboring cells
(arrows) will eventually move to cover the cleft. (G) Ventral view
showing epiboly of the skin cells (arrows) to cover remaining cells
on the ventral surface.
localized exclusively to sites of cell contact in a wild-type 4cell embryo (Costa et al., 1998), a pattern very similar to that
of PAR-2. However hmr-1(RNAi) embryos, with markedly
depleted HMR-1 levels, had the wild-type pattern of both PAR-
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J. Nance and J. R. Priess
2 and PAR-3 localization (data not shown). We next asked if
the apical-basal polarity in PAR localization was dependent on
par gene function. As reported previously by others (Boyd et
al., 1996; Etemad-Moghadam et al., 1995), we observed that
PAR-2 was not restricted to the posterior pole in 1-cell stage
par-3 mutant embryos, and that PAR-3 was not restricted to
the anterior pole in par-2 mutant embryos. During the 2-cell
to 16-cell stages, PAR-2 was not restricted to basal and lateral
surfaces in par-3 mutant embryos, and was instead associated
with the entire cortex of each blastomere (Fig. 2H) (see also
Boyd et al., 1996). PAR-3 showed the wild-type pattern of
apical localization in par-2 mutant embryos (n=40), but failed
to accumulate at the cortex in par-6 mutants (n=15). We
conclude that PAR-3 is required to exclude PAR-2 from the
apical surface. PAR-3 localization to the apical surface appears
to be independent of both HMR-1 and PAR-2, but requires
PAR-6.
Ingression of cells during gastrulation
Ingression occurs on the ventral surface of the embryo. Many
of the cells that ingress are born on the ventral surface (E, MS
and P4 descendants; Fig. 4B). Other cells, such as the C
descendants (green cells in Fig. 4B), are born in dorsal or
lateral positions and move to the ventral surface prior to
ingression. We analyzed 4D videorecordings of gastrulating
embryos to determine when and where cells ingress. Cells do
not ingress from a single location, but rather enter the
blastocoel from several positions over the ventral surface of the
embryo; these patterns of ingression are summarized in Fig.
4C, and some of the major groups of ingressing cells are
described here briefly.
The daughters of the E blastomere are the first to ingress,
beginning at the 26-cell stage (90 minutes after the first
embryonic division). Ingression of the E daughters (yellow
asterisks in Fig. 4D) takes about 16 minutes, and creates a
transient opening on the surface of the embryo. This opening
is sealed as neighboring cells, primarily MS descendants and
P4 (red and cyan arrowheads, respectively, Fig. 4D), spread
across the surface of the E daughters. In scanning electron
micrographs of the ventral surface, the MS descendants show
wedge-shaped processes that are oriented toward the ingressing
E daughters (Fig. 5A,B). The leading edges of these processes
taper into thin sheets over the surfaces of the E daughters (Fig.
5C). Although the P4 blastomere moves across the surfaces of
the E daughters, it does not extend processes resembling those
from the MS descendants (Fig. 5).
By 150 minutes most of the ventral surface of the embryo
is occupied by the 16 descendants of the MS blastomere. 12
of the MS descendants ingress at about 150 minutes (red
‘wishbone’ shape in Fig. 4C). The 4 remaining MS
descendants stay on the surface of the embryo for an additional
cell cycle before ingressing at about 190 minutes (red oval
shape in Fig. 4C, and red asterisks in Fig. 4E). The ingression
of the ‘wishbone’ group of 12 MS descendants, as well as the
ingression of the P4 descendants, each creates a transient
surface gap that is quickly enclosed by neighboring cells. In
contrast, the surface gaps created by ingression of the D
descendants (arrow, Fig. 4E) and by ingression of the
remaining MS descendants (arrows, Fig. 4F) persist for 20-30
minutes. In all cases examined, sister cells ingressed either
simultaneously or within a few minutes of each other (see
Fig. 5. Cell extensions over
ingressing cells.
(A) Scanning electron
micrograph of the ventral
surface of a 28-cell embryo,
anterior is to the left.
(B) Tracing of A to indicate
cell names. The exposed
apical surfaces of the
ingressing E daughters are
tinted. (C) High
magnification of the region
corresponding to the boxed
area in B. The arrow indicates
the thin, leading process from
an MS descendant.
Discussion). After the cycles of ingression are completed, the
ventral surface of the embryo is occupied primarily by
neuronal precursors. These precursors do not appear to enter
the blastocoel by ingression, but are internalized by epiboly of
skin cells [Fig. 4G; reviewed by Simske and Hardin (Simske
and Hardin, 2001); see also Sulston et al. (Sulston et al.,
1983)].
Control of cell ingression
Cells in the left half of the ‘wishbone’ group of MS
descendants ingress at the same time and have the same fate
as cells in the right half of the wishbone [see Fig. 4C; see also
Sulston et al. (Sulston et al., 1983)]. The observation that cells
with identical fates, but with different positions, can ingress at
the same time suggests that cell fate could determine the time
of ingression. In their analysis of the C. elegans cell lineage,
Sulston et al. (Sulston et al., 1983) documented several
examples of pairs of cells that were located in different parts
of the embryo but that had identical, or nearly identical,
patterns of division and differentiation. We selected nine such
examples of AB descendants (cell pairs numbered a and b
in Table 1), and determined their ingression times from
videorecordings of live embryos. Cells with the same fate
usually had remarkably similar ingression times (8/9 cases).
For example, the ingression times of cells 4a and 4b differed
by only 1 minute (Table 1). In contrast, cells that were born in
equivalent cell cycles, but that had different fates could differ
significantly in ingression times. For example, the ingression
times of cells 4a and 9b differed by 77 minutes (Table 1).
Patterning ingressions in MS descendants
If cell fate, rather than position, is the sole determinant of
ingression, mutations that affect cell fate should alter the
pattern of ingression. Mutations in the mex-1 gene cause AB
descendants to express a transcription factor that normally
C. elegans polarity and gastrulation
393
Table 1. Difference in ingression times of AB descendants
Pair*
Time (minutes)‡
1b
2b
3b
4b
5b
6b
7b
8b
9b
193
221
217
212
269
232
245
296
288
1a
193
0
28
24
19
76
39
52
103
95
2a
212
19
9
5
0
57
20
33
84
76
3a
210
17
11
7
2
59
22
35
86
78
4a
211
18
10
6
1
58
21
34
85
77
5a
238
45
17
21
26
31§
6
7
58
50
6a
241
48
20
24
29
28
9
4
55
47
7a
243
50
22
26
31
26
11
2
53
45
8a
287
94
66
70
75
18
55
42
9
1
9a
288
95
67
71
76
19
56
43
8
0
*Each number/letter combination represents an AB descendant; pairs of descendants with the same number prefix have similar lineages. Pairs 1a,b and 2a,b
ingress in the 8th AB cell cycle while all other pairs ingress in the 9th AB cell cycle. 1a,b: ABaraapaa, ABaraappa; 2a,b: ABalpaaap, ABarapaap; 3a,b:
ABalpapppp, ABarapappp; 4a,b: ABalpapppa, ABarapappa; 5a,b: ABalpappaa, ABarapapaa; 6a,b: ABalpaapap, ABaraaapap; 7a,b: ABalpaapaa, ABaraaapaa;
8a,b: ABalpaaaap, ABarapaaap; 9a,b: ABalpaaaaa, ABarapaaaa.
‡Time when ingression initiates (in minutes from the 2-cell stage). Values listed in the remainder of the matrix are differences between these times. Differences
among pairs 1 and 2, which ingress one cell cycle earlier than all other pairs, are boxed.
§Although the ancestry and subsequent differentiation of cell 5b is most similar to cell 5a, several AB descendants share the identical ‘sublineage’ (Sulston et
al., 1983).
specifies the MS fate (Bowerman et al., 1993; Mello et al.,
1992). We found that several AB descendants that do not
ingress in wild-type embryos did ingress in mex-1 mutant
embryos analyzed over the period corresponding to normal MS
ingression (n=2). However, many of the descendants of the
transformed AB cells did not ingress, in contrast to wild-type
MS descendants, and the pattern of ingression varied between
each of the mex-1 embryos.
To understand how ingression times are determined in the
wild-type MS lineage, we focused on the difference in
ingression times between the MS ‘wishbone’ descendants and
their closely related relatives in the center of the wishbone
(hereafter called the central cells; see Fig. 4C). As described
above, the MS central cells (asterisks, Fig. 6A) remain on the
surface for one cell cycle after the wishbone cells ingress. The
central cells that remain on the surface overlie E descendants
in the center of the embryo, while the wishbone cells ingress
along the perimeter of the E descendants. We therefore asked
whether preventing E ingression would allow the central cells
to ingress with the wishbone cells. Although killing the E
blastomere with a laser microbeam prevented ingression of the
E daughters, the central cells did not ingress along with the
wishbone cells (12/12 embryos, Fig. 6B). We obtained
identical results in experiments where the fate of the E
blastomere was altered by a chromosomal deficiency of the
endoderm determining region (Zhu et al., 1997); although the
E daughters and many of the subsequent E descendants
remained on the surface of the mutant embryos, the wishbone
and central cells showed the wild-type difference in ingression
times (6/6 embryos).
By lineage, the central cells are posterior relatives of the
anterior wishbone cells. In several cases studied, anteriorposterior differences between closely related cells in C. elegans
are determined by anterior-posterior asymmetry in the
expression of the transcription factor POP-1 (Lin et al., 1998;
Lin et al., 1995), and POP-1 asymmetry can be controlled by
the kinase LIT-1 (Kaletta et al., 1997; Meneghini et al., 1999;
Rocheleau et al., 1999). We therefore shifted embryos
homozygous for the temperature-sensitive mutation lit1(t1512ts) to the restrictive temperature before the birth of the
central cells; this temperature shift should have caused the
central cells to have the same pattern of POP-1 expression as
the wishbone cells. However, the central cells did not ingress
at the same time as the wishbone cells in any of these
experiments (n=7; Fig. 6C). We next asked whether the
presence of the E descendants in the blastocoel might block
ingression of the central cells by killing the E blastomere in
temperature-shifted lit-1 mutant embryos. In 4 of 6 embryos
Fig. 6. Control of MS ingression. All panels are ventral views of
embryos after ingression by the MS wishbone descendants; the MS
central descendants are indicated by asterisks. The central
descendants remain on the surface in (A) a normal embryo, (B) an
embryo after ablation of the E blastomere (dotted outline) and (C) a
temperature-shifted lit-1 mutant embryo. (D) The central
descendants ingress (arrow) along with the wishbone descendants in
a temperature-shifted lit-1 mutant embryo after ablation of the E
blastomere (dotted outline). The lit-1 chromosome was marked with
an unc-32 mutation; the embryo shown in B is a temperature-shifted
unc-32 control.
394
J. Nance and J. R. Priess
Schierenberg (Junkersdorf and Schierenberg, 1992)],
suggesting apical flattening is a cell-autonomous process.
Cell shape changes often are driven by actin and non-muscle
myosin. We therefore examined the distribution of NMY-2, a
C. elegans non-muscle myosin (Guo and Kemphues, 1996).
NMY-2 is present at low levels at the cell cortex during all
stages of the cell cycle (Fig. 7C,D), and is strongly enriched in
the contractile ring or midbody of mitotic cells (arrows, Fig.
7C,D). NMY-2 began to show enrichment on the apical
surfaces of the E daughters just prior to ingression (24-cell
stage, 6/36 embryos). This enrichment was evident in most E
daughters at the onset of ingression (26-cell stage, 36/43
embryos; Fig. 7D), and in all E daughters near the end of
ingression (28-cell stage, 20/20 embryos. The level of NMY-2
at the apical surface decreased once the E daughters entered
the blastocoel (data not shown). We observed a similar apical
enrichment of NMY-2 on ingressing MS descendants
(arrowhead, Fig. 7F).
DISCUSSION
Fig. 7. Cell-shape changes in ingressing cells. Images are of living
embryos (A,B,E) or are confocal sections of fixed embryos after
immunostaining for NMY-2 (C,D,F). (C,D,F) Confocal sections. All
embryos are oriented as in Fig. 4D, and arrowheads indicate apical
surfaces. (A-D) The E daughters are indicated (yellow asterisks)
before ingression (A,C) and during ingression (B,D).
(E-F) Ingression of MS descendants (red asterisks). MSapaa is
shown in (E) and two of the MS ‘wishbone’ descendants are shown
in (F), in which NMY-2 is green and DNA blue. A,B,E and F are
shown at twice the magnification of C and D.
tested, the central cells ingressed at the same time as the
wishbone cells (arrow, Fig. 6D). Because the lit-1(t1512ts)
allele is incompletely penetrant, we consider it likely that POP1 expression was not altered in the two embryos with normal
ingression times (see Kaletta et al., 1997). We conclude that
the ingression pattern of the MS central descendants is
controlled by redundant mechanisms involving cell fate
specification and interactions with E descendants.
Cell shape changes in ingressing cells
In living embryos, the apical surfaces of the E daughters
initially appear rounded (arrowhead, Fig. 7A), similar to the
apical surfaces of neighboring cells. Shortly before ingression,
their apical surfaces flatten (Fig. 7B), and remain flattened
during ingression; small, transient protrusions of the apical
membranes appear during flattening (data not shown). We
observed a similar apical flattening during ingression of the MS
descendants (Fig. 7E), suggesting that apical flattening is a
general characteristic of ingressing cells.
Apical flattening during ingression could be autonomous or
a response to tension generated by neighboring cells. In normal
development, the MS descendants extend processes over the E
daughters during ingression (see Fig. 5). However, we found
that the E daughters underwent apical flattening and ingressed
even after the MS blastomere was killed with a laser
microbeam [10/10 embryos; see also Junkersdorf and
Embryo architecture and strategies of gastrulation
In comparison to other animal systems (see Introduction),
gastrulation in C. elegans involves both cell ingression and
epiboly. Embryogenesis occurs within a fixed space limited by
the eggshell, and the volume of the blastocoel is very small,
less than the volume of a single cell at the onset of gastrulation.
Thus the first cell ingressions must be coupled to simultaneous
redistributions in the masses of other embryonic cells. During
ingression of the E daughters, this redistribution appears to be
accomplished primarily by the MS descendants that
immediately spread across the site of ingression. At later
embryonic stages the ingressing cells are relatively small and
there is less need for the surrounding cells to redistribute their
mass; at these stages surface gaps persist over the sites of
ingression for long periods of time.
Gastrulation in embryos like Drosophila involves sheets of
cells that invaginate to form furrows and pockets. While
adherens junctions link the surface cells of Drosophila
embryos at the time of gastrulation (Oda et al., 1998; Tepass
and Hartenstein, 1994), adherens junctions are not visible in C.
elegans embryos until most of the cell movements associated
with gastrulation are complete (Costa et al., 1998) (our
unpublished observations). The absence of adherens junctions
in early C. elegans embryos may thus facilitate ingression by
small groups of cells. In sea urchins, primary mesenchyme
cells appear to lose adherens junction connections with
neighboring surface cells during ingression (Miller and
McClay, 1997a; Miller and McClay, 1997b). In our survey of
ingression times in C. elegans embryos, we noted that sister
cells ingressed at approximately the same time; solitary cells
did not ingress. A likely possibility is that the midbody linkage
between sister cells prevents either sister from ingressing
alone. Both electron microscopic and immunocytochemical
studies have shown that the embryonic cells in C. elegans
remain coupled by midbodies to their sisters through most of
the cell cycle (Krieg et al., 1978) (J. N. and J. R. P., unpublished
observations).
Ingressing cells in C. elegans embryos show an apical
flattening and an apical accumulation of non-muscle myosin.
C. elegans polarity and gastrulation
Apical flattening could promote ingression by redistributing
cytoplasm toward the basal region of an ingressing cell, and
create space at the surface of the embryo for neighboring cells
to redistribute their cytoplasm laterally. The invagination of
epithelial sheets in Drosophila gastrulation is also associated
with an apical flattening and accumulation of non-muscle
myosin (Leptin and Grunewald, 1990; Sweeton et al., 1991;
Young et al., 1991). Thus the cellular basis for invagination by
epithelial sheets and for ingression by small groups of cells
may be conserved, with the principal difference being whether
the cells are linked by junctional complexes.
Cell fate and ingression
In principle, gastrulation could position a subset of unspecified
cells into the interior of the embryo, where subsequent
events would specify the endodermal and mesodermal fates
appropriate for internal organs. Instead, C. elegans and many
other animals appear to specify the fates of endodermal and
mesodermal progenitors when these progenitors are on the
embryo surface. Thus there must be direct or indirect
mechanisms that couple gastrulation movements with cell fate.
Our present study provides evidence that cell fate is tightly
linked to cell ingression in C. elegans. For example, mex-1
mutants with ectopic MS-like cells show ectopic ingressions
during the period MS descendants normally ingress, and a
mutation in lit-1 that changes the fate of MS descendants can
change the ingression time of those descendants. Similarly, in
Drosophila the cell fate regulators snail and twist appear to
control invagination (reviewed by Leptin, 1995).
The molecular connections between cell fate and ingression
or invagination are not known. Embryonic transcription is
required for gastrulation in C. elegans (Powell-Coffman et al.,
1996), though we have shown here that transcription is not
required for the apical-basal localization of the PAR proteins
or for blastocoel formation. Instead, embryonic transcription is
required for the apical concentration of non-muscle myosin in
ingressing cells and for the apical flattening of these cells. A
simple model is that the cellular machinery required for
ingression or invagination is regulated by one master control
gene, and that this gene is in turn regulated by any one of
several embryonically expressed transcription factors that
specify cell fate. By analogy, the Drosophila cell cycle
regulator string has a large, complex promoter controlled by
numerous transcription factors that also control region-specific
differentiation, creating regions of mitosis that match regions
of common cell fate (Edgar et al., 1994; Johnston and Edgar,
1998; Lehman et al., 1999).
The control of MS ingression in C. elegans appears to be an
example of fine-tuning in a developmental system. After
ingression of the E daughters, the largest available spaces in
the blastocoel are anterior to, and on both sides of, the E
daughters. The MS wishbone cells overlie these spaces, and
ingress into them. In contrast, the MS central cells overlie the
E daughters, and do not ingress at the same time as the
wishbone cells. Our analysis of lit-1 mutant embryos
demonstrates that the presence of the E daughters in the
blastocoel is sufficient to prevent ingression of the central cells
during ingression of the wishbone cells; we consider it likely
that this is a steric interaction, but cannot exclude more specific
cell signaling. However, the MS central cells are subject to an
additional, lit-1-dependent, control that prevents ingression
395
even when space in the blastocoel is made available by killing
the E blastomere. We propose that in normal development this
mechanism prevents the central cells from attempting to
ingress when there is not sufficient space to do so.
Establishing apical-basal polarity and the PAR
proteins
The events of gastrulation indicate that early embryonic cells
have apical-basal polarity; for example, NMY-2 accumulates
at the apical surface of ingressing cells, and the blastocoel
forms at the basal surfaces. Apical-basal asymmetry is evident
in embryonic cells of C. elegans as early as the 4-cell stage:
PAR-3 and PAR-6 are localized to the apical surfaces, and
PAR-2 and HMR-1(cadherin) are localized to the basal and
lateral surfaces of cells. Drosophila and vertebrate homologues
of PAR-3 and PAR-6, together with the atypical protein kinase
PKC-3, have been shown to function in establishing apicalbasal polarity in epithelial cells and neuroblasts (reviewed by
Ohno, 2001). We have shown here that large cell separations
are observed between both basal and lateral surfaces of cells
in par-3 mutants. This result suggests that par-3(+) may have
a role in distinguishing the basal from lateral surfaces during
blastocoel formation in wild-type embryos, where large
separations only occur between basal surfaces.
We have shown that cell contacts restrict PAR-3 and PAR-6
to the contact-free, apical surfaces. This mechanism differs, at
least in part, from the mechanism that localizes these same
proteins to the anterior surface of the 1-cell embryo. While
PAR-2 has a role in determining PAR-3 localization at the 1cell stage, PAR-2 is not required for the apical localization of
PAR-3 at the 4-cell stage, nor is it required for blastocoel
formation. HMR-1(cadherin) also is localized to cell contacts,
but does not appear to have a role in PAR localization or
blastocoel formation (our present results) (Costa et al., 1998).
Interestingly, genetic or immunological inhibition of Ecadherin function in early mouse embryos does not prevent
individual cells from becoming polarized, but rather causes a
randomization in the axis of polarity (Johnson et al., 1986;
Larue et al., 1994; Riethmacher et al., 1995). HMR-1 appears
to be the only ‘classical’ cadherin with a β-catenin binding site,
similar to mouse E-cadherin (Hill et al., 2001), although the C.
elegans genome sequence predicts several cadherin-related
proteins whose functions and localization have not been
determined (Hill et al., 2001).
Localization of PAR-3, or associated proteins, to the apical
surface could in principle differentiate the basal surface from
the lateral surface. For example, the localization of ion
channels to the apical surface could create a gradient that
affects the opposite, basal, surface differently than the lateral
surfaces. Vectorial ion transport is essential for formation of
the blastocoel in mouse embryos, and channel proteins appear
to be localized with apical-basal polarity in trophectodermal
cells lining the blastocoel [reviewed by Watson and Barcroft
(Watson and Barcroft, 2001)]. It will be interesting in future
studies to determine how apical-basal polarity of the PAR
proteins directs subsequent asymmetries. In the 1-cell embryo,
the anterior-posterior polarity of the PAR proteins establishes
a parallel gradient of MEX-5 in the cytoplasm. MEX-5 in turn
functions to prevent the anterior expression of posterior
proteins (Schubert et al., 2000). However, MEX-5 is uniformly
distributed in somatic blastomeres that have an apical-basal
396
J. Nance and J. R. Priess
polarity in PAR protein distribution, suggesting that MEX-5
does not mediate apical-basal polarity.
We thank Judith Austin, Ken Kemphues, Susan Mango and
Geraldine Seydoux for providing transgenic strains; Ichiro Kawasaki
and Susan Strome for PGL-1 antibodies; and Kathryn Baker and Ken
Kemphues for the extremely generous gifts of PAR and NMY-2
antibodies. We thank Judy Groombridge and Franque Remington for
assistance with electron microscopy, Jason Pitt for providing advice
and Nematode Navigator software, and members of the Priess
laboratory for useful discussions and suggestions. Some of the
nematode strains used in this study were provided by the
Caenorhabditis Genetics Center, which is funded by the NIH National
Center for Research Resources (NCRR). J.N. and J.R.P. are supported
by the Howard Hughes Medical Institute.
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