3815
Development 124, 3815-3826 (1997)
Printed in Great Britain © The Company of Biologists Limited 1997
DEV5136
The maternal par genes and the segregation of cell fate specification
activities in early Caenorhabditis elegans embryos
Bruce Bowerman1,*, Malene K. Ingram1,‡ and Craig P. Hunter2,†
1Institute of Molecular Biology,
2Department of Molecular and
University of Oregon, 1370 Franklin Blvd, Eugene, Oregon 97403, USA
Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA
*Author for correspondence (e-mail: [email protected])
†Author for correspondence (e-mail: [email protected])
‡Present address: College of Human Medicine, Michigan State University, 226W Owen Graduate Center, East Lansing, MI 48825, USA
SUMMARY
After fertilization in C. elegans, activities encoded by the
maternally expressed par genes appear to establish cellular
and embryonic polarity. Loss-of-function mutations in the
par genes disrupt anterior-posterior (a-p) asymmetries in
early embryos and result in highly abnormal patterns of
cell fate. Little is known about how the early asymmetry
defects are related to the cell fate patterning defects in par
mutant embryos, or about how the par gene products affect
the localization and activities of developmental regulators
known to specify the cell fate patterns made by individual
blastomeres. Examples of such regulators of blastomere
identity include the maternal proteins MEX-3 and GLP-1,
expressed at high levels anteriorly, and SKN-1 and PAL-1,
expressed at high levels posteriorly in early embryos. To
better define par gene functions, we examined the
expression patterns of MEX-3, PAL-1 and SKN-1, and we
INTRODUCTION
The generation of asymmetry before mitosis is a general
mechanism for producing cells with different fates during
growth and development (for a review, see Gonczy and Hyman,
1996). For example, sperm entry provides an extrinsic cue to
polarize the 1-cell zygote in C. elegans, defining the anteriorposterior (a-p) body axis (Hird and White, 1993; Goldstein and
Hird, 1996). Polarization of the nematode zygote results in
posterior displacement of the first mitotic spindle, producing a
smaller P1 and a larger AB daughter, two cells with very
different fates (Sulston et al., 1983). AB and P1 differ in their
cell cycle times and in the orientation of their mitotic spindles:
AB divides first with a transversely oriented spindle to make
two daughters of equal size, while P1 divides after AB, with its
spindle aligned parallel to the long axis and displaced posteriorly, dividing unequally (Hyman and White, 1987; Hyman,
1989; see Fig. 1). The different developmental potentials of P1
and AB also are apparent when each is cultured in isolation:
only P1 and not AB can produce pharyngeal cells, intestinal
cells and body wall muscle cells (Laufer et al., 1980; Priess
and Thomson, 1987; Draper et al., 1996). Consistent with their
analyzed mex-3, pal-1, skn-1 and glp-1 activities in par
mutant embryos. We have found that mutational inactivation of each par gene results in a unique phenotype, but in
no case do we observe a complete loss of a-p asymmetry.
We conclude that no one par gene is required for all a-p
asymmetry and we suggest that, in some cases, the par
genes act independently of each other to control cell fate
patterning and polarity. Finally, we discuss the implications
of our findings for understanding how the initial establishment of polarity in the zygote by the par gene products
leads to the proper localization of more specifically acting
regulators of blastomere identity.
Key words: anterior/posterior asymmetry, cell fate specification, cell
polarity, cortical cytoplasm, glp-1, mex-3, pal-1, par-1, par-2, par-3,
par-4, skn-1
different developmental potentials, genetically identified regulators of pattern formation are differentially localized to P1 and
AB, and their descendants (see Fig, 1).
Of the maternal genes known to control pattern formation in
C. elegans, mutations in six, par-1 through par-6, cause the
earliest and most extensive polarity defects in the zygote, eliminating many early asymmetries (Kemphues et al., 1988; Kirby
et al., 1990; Morton et al., 1992; Cheng et al., 1995; Guo and
Kemphues, 1996; Kemphues and Strome, 1997). For example,
in all par mutant embryos P1 and AB divide synchronously, and
they are of equal size in all but par-4 mutant embryos. Furthermore, of the four PAR proteins examined thus far, all are
present at low levels throughout the cytoplasm and at much
higher levels in the cytoplasmic cortex, with the cortical enrichment being polarized along the a-p axis for PAR-1, PAR-2 and
PAR-3 (Levitan et al., 1994; Etemad-Moghadam and
Kemphues, 1995; Guo and Kemphues, 1995; Boyd et al., 1996);
J. Watts, D. Morton and K. Kemphues, personal communication). Initially, PAR-1 and PAR-2 are present cortically throughout mature oocytes but, after fertilization, both are detected only
in the posterior cortex of the 1-cell zygote, called P0 (Guo and
Kemphues, 1995; Boyd et al., 1996; see Fig. 1). Complemen-
3816 B. Bowerman, M. K. Ingram and C. P. Hunter
tary to PAR-1 and PAR-2, cortical PAR-3 is present only in the
anterior of P0, and the posterior boundary of the cortical PAR3 in P0 abutts the anterior boundary of PAR-1 and PAR-2
(Etemad-Moghadam and Kemphues, 1995; Kemphues and
Strome, 1997; see Fig. 1). PAR-4 is unique in that although
enriched cortically it is not polarized along the a-p axis (J.
Watts, D. Morton, and K. Kemphues, personal communication).
While four par genes have been identified molecularly, how
they establish or regulate polarity remains almost entirely
unkown. PAR-1 contains a predicted N-terminal ser/thr kinase
domain and a C-terminal domain that interacts with a nonmuscle conventional myosin (Guo and Kemphues, 1996a).
PAR-2 contains a putative ATP-binding site and a zinc-binding
domain of the ‘RING finger’ class (Levitan et al., 1994). PAR3 is a novel protein with three PDZ repeats, and PAR-4
contains a ser/thr kinase domain (Etemad-Moghadam and
Kemphues, 1995); J. Watts and K. Kemphues, personal communication). Intriguingly, PAR-3 extends posteriorly in par-2
mutant embryos, and PAR-2 extends anteriorly in par-3
mutants, suggesting that par-2 and par-3 interact during the
specification of a-p polarity (Etemad-Moghadam and
Kemphues, 1995; Boyd et al., 1996). Indicating another interaction, PAR-1 is not enriched cortically in par-2 mutant
embryos (Guo and Kemphues, 1995). Because par mutant
embryos exhibit very early defects in polarization of the
zygote, and because the PAR proteins themselves become
asymmetrically distributed shortly after fertilization, the par
genes likely encode machinery that polarizes the zygote
directly in response to sperm entry, initiating processes that
ultimately localize more specifically acting regulatory factors
to individual blastomeres (Guo and Kemphues, 1996b;
Kemphues and Strome, 1997). Homologs of par-1 called
MARK kinases have been identified in yeast and in mammals
(Levin et al., 1987; Levin and Bishop, 1990; Drewes et al.,
1997). The involvement of these closely related kinases in
regulating polarity in yeast and microtubule stability in
mammalian cells indicates that understanding par gene
functions in C. elegans is of general importance.
To better understand the par genes, we investigated how
mutations in par-1, par-2, par-3 and par-4 affect the function
of four other maternally expressed gene products encoded by
skn-1, glp-1, pal-1 and mex-3. Mutations in these latter genes
cause defects in the fates of individual blastomeres without
causing more general defects in polarity characteristic of par
mutants. The skn-1 gene encodes a putative transcription factor
required to specify the fate of one P1 daughter, EMS
(Bowerman et al., 1992, 1993; Blackwell et al., 1994). skn-1
also activates a signal that induces ABa descendants to produce
pharyngeal cells (Shelton and Bowerman, 1996), and glp-1
encodes a Notch-like receptor required for ABa descendants to
respond to this signal (Priess and Thomson, 1987; Austin and
Kimble, 1989; Yochem and Greenwald, 1989; Evans et al.,
1994; Fig. 1). PAL-1 is a homeodomain protein and specifies
the production of body wall muscle and epidermis by the P2
daughter of P1 (Hunter and Kenyon, 1996). MEX-3 is a
putative RNA-binding protein required to prevent translation
of pal-1 mRNA in anterior blastomeres in the early embryo
(Draper et al., 1996; Hunter and Kenyon, 1996).
By examining the expression and function of SKN-1, GLP1, PAL-1 and MEX-3, we show that par-1, par-2 and par-3
mutant embryos, although superficially similar in terminal
phenotype, each exhibit a unique set of cell fate patterning
defects. par-4 mutant embryos show even less phenotypic similarity to the other par mutants with respect to the developmental pathways that we analyze. These results indicate that
the par genes have substantially distinct roles in segregating
different cell fate specification activities. Therefore we think it
unlikely that the par genes operate in a single pathway. Instead,
we suggest that the par genes encode part of a possibly
complex network of interacting gene products that link polarization in the zygote to the patterning of specific blastomere
identities.
MATERIALS AND METHODS
Strains and alleles
Nematode cultures were maintained as described (Brenner, 1974).
The genotypes of strains used for analysis of mutant phenotypes were
as follows: +/DnT1 (IV;V) IV; par-1(e2012) rol-4(sc8)/DnT1(IV;V) V,
skn-1(zu67)/DnT1(IV;V) IV; par-1(e2012) rol-4(sc8)/DnT1(IV;V),
par-2(lw32) unc-45(e286ts)/sC1[dpy-1(e1) let] III, par-2(lw32) unc45(e286ts)/sC1[dpy-1(e1)
let]
III;
dpy-13(e184sd)
skn1(zu67)/nT1(IV;V) IV; +/nT1(IV;V) V, par-2(lw32) dpy-1(e1) unc32(e189) glp-1(e2142ts)/ dpy-17(e224) unc-32(e189) glp-1(e2142ts)
III, par-3(it71) lon-1(e185)/qC1[dpy-19(e1259ts) glp-1(q339)] III,
par-3(it71) lon-1(e185) unc-32(e189) glp-1(e2142ts)/ dpy-17(e164)
unc-32(e189) glp-1(e2142ts) III, lon-1(e185) par-3(it71)/qC1[dpy19(e1259ts) glp-1(q339)] III; skn-1(zu67)/DnT1(IV;V) IV;
+/DnT1(IV;V)V, par-4(it47ts) V, glp-1(e2142ts) unc-32(e189) III; par4(it47ts) V, mDp1 (f,IV); unc-5(e53) skn-1(zu67) IV; par-4(it57ts) V.
All glp-1 strains and par-4 strains were maintained at 15°C and adults
shifted to 25°C 2 hours before collection of embryos. DpyUnc par-2
glp-1 mothers were identified because they were more severely Dpy
than DpyUnc glp-1 siblings. In all ablation experiments, the presence
of par-2 or par-3 were confirmed by using only those embryos in
which P1 and AB were of equal size and by confirming the phenotype
of unablated siblings using Nomarski optics to score characteristic cell
types before transferring partial embryos for antibody staining. In
ablations using par-4(it47ts), mutant embryos were identified by
waiting to score the synchrony of the P1 and AB cleavages.
The skn-1 allele zu67 is the strongest skn-1 allele and corresponds
to a premature stop codon in the second coding exon (C. Schubert, B.
Bowerman and J. Priess, unpublished data). At 25°C, the glp-1 allele
e2142ts specifically eliminates the ability of GLP-1 to respond to the
12-cell-stage MS induction of pharyngeal cell production by ABa
descendants, without affecting the response to an earlier signal from
P2 (Hutter and Schnabel, 1994; Mello et al., 1994). par-1(e2012) is
the most penetrant par-1 allele identified with respect to the lack of
intestinal cells (Kemphues et al., 1988). par-2(lw32) is a strong allele
with a nonsense mutation predicted to encode 233 of 628 predicted
amino acids (Levitan et al., 1994). par-3(it71) appears to be a protein
null (Cheng et al., 1995; Etemad-Moghadam and Kemphues, 1995).
The par-4 allele it57ts is a strong allele when grown at 25°C (Cheng
et al., 1995).
Embryo manipulations and microscopy
1-cell and 2-cell wild-type and mutant embryos were collected by cutting
open gravid adults in a watch glass filled with M9 buffer, mounted on
3% agarose pads under a coverslip as described (Sulston et al., 1983).
Blastomeres were killed using 10-25 pulses from a Laser Science, Inc.,
VSL-337 laser microbeam directed into a Zeiss Axioskop (Avery and
Horvitz, 1989). Partial embryos were allowed to develop overnight for
15-20 hours at room temperature for analysis of pharyngeal muscle cell
production, or for 8-10 hours at room temperature for analysis of body
wall muscle cell production. Antibody staining procedures for pharyngeal muscle and body wall muscle, and for SKN-1, PAL-1 and MEX-3
Patterning in par mutants 3817
were as described (Bowerman et al., 1993; Draper et al., 1996; Hunter
and Kenyon, 1996). Intestinal cell-specific gut granules were scored
using polarizing optics (Bowerman et al., 1992). For analysis of
unablated embryos, homozygous mutant mothers were collected and
either allowed to lay embryos for several hours or cut open to obtain
embryos; the embryos were then allowed to develop and analyzed for
pharyngeal muscle and body wall muscle cells as for partial embryos.
We used Kodak Technical Pan film and HC110 developer, a Polaroid
Sprint Scan 35 and Adobe Photoshop Version 4.0 for making figures.
RESULTS
Specification of pharyngeal cell fates in the early
embryo
To define requirements for the par genes in segregating cell
fate specification activities, we first examined the patterning of
pharyngeal cell fates in par mutant embryos. Wild-type
embryos produce 37 pharyngeal muscle cells, 19 mostly
anterior muscles derived from ABa and 18 mostly posterior
muscles from MS (Sulston et al., 1983; see Fig. 2A). As illustrated in Fig. 1, two granddaughters of ABa require glp-1
function to produce pharyngeal cells in response to an
inductive signal from MS at about the 12-cell stage (Priess et
al., 1987; Mango et al., 1994; Hutter and Schnabel, 1995).
Consequently, in glp-1 mutant embryos only MS produces
pharyngeal muscle cells (Priess et al., 1987; see Fig. 2B). skn1 function is required both for specifying pharyngeal cell production by MS and for activating the signal from MS that
induces production of ABa-derived pharyngeal cells
(Bowerman et al., 1992; Shelton and Bowerman, 1996). Consequently skn-1 mutant embryos fail to produce any pharyngeal cells (Fig. 2C). The requirements for the par genes in
restricting pharyngeal cell specification activities to individual
blastomeres have been examined most extensively in par-1
mutant embryos (Bowerman et al., 1993; Draper et al., 1996;
Hunter and Kenyon, 1996). Here, we report an analysis of glp1 and skn-1 function in par-2, par-3 and par-4 mutant embryos.
Pharyngeal cell fate specification pathways in par-2,
par-3 and par-4 mutant embryos
To analyze the activities of skn-1 and glp-1 in par mutant
embryos, we examined the patterning of pharyngeal cell fates
in par mutant embryos in which either glp-1 or skn-1 activity
was absent (Fig. 2). As shown previously for par-1 mutants,
we have found that most par-2 and par-3 mutants produce a
large excess of pharyngeal muscle cells in both the anterior and
posterior portions of the embryo (Fig. 2G,J). The pharyngeal
phenotype of par-4 mutant embryos is more variable: large
numbers of pharyngeal muscle cells are present posteriorly in
about 50% of par-4 mutant embryos (Fig. 2M), but no pharyngeal cells are made in the remaining 50% (Fig. 2 legend).
As expected, all par mutant embryos require skn-1 function to
produce any pharyngeal cells (Fig. 2F,I,L,O). We next asked if
the production of pharynx was dependent on glp-1 activity. We
found that par-3; glp-1 and par-4; glp-1 double mutant
embryos produce indistinguishable numbers and distributions
of pharyngeal cells compared to par-3 and par-4 single mutants
(compare Fig. 2J and K, M and N). In contrast, par-2; glp-1
double mutant embryos produce substantially fewer pharyngeal cells than do par-2 mutant embryos (compare Fig. 2G and
H). Thus par-2 mutant embryos are unique in producing glp1-dependent pharynx anteriorly. In par-1, par-3 and par-4
anterior
dorsal
posterior
AB
p
D
A
P2
P0
ABa
EMS
ventral
B
C
E
P3
E
C
MS
AB
Key:
P1
PAR-1, PAR-2
SKN-1
PAR-3
PAL-1
MEX-3
(high levels)
MEX-3
(low levels)
GLP-1
Fig. 1. Schematic of polarization and patterning in the early C.
elegans embryo. Blastomere names (P0, P1, AB, P2, EMS, ABa,
ABp, P3, C, E and MS) are adjacent to the corresponding cells.
(A) Early 1-cell-stage embryo, called P0, after fertilization. The
maternal pronucleus is at the left end of the zygote, and the paternal
pronucleus, with an associated centriole, is at the right. Sperm entry
typically occurs opposite the maternal pronucleus. After fertilization,
the maternal pronucleus completes meiosis, producing two polar
bodies (small open circle). After completion of meiosis, an actindependent cytoplasmic flux occurs (curved and dashed arrows).
(B) Dividing 1-cell-stage embryo, with the first mitotic spindle
aligned on the long axis and displaced posteriorly. The polarized
distributions of PAR-1 and PAR-2 (brown), and PAR-3 (tan), are
inherited by the P0 descendants P1, P2 and P3, precursors to the final
germline progenitor P4. For simplification, the PAR distributions are
shown only at the 1-cell stage. (C) Dividing 2-cell-stage embryo,
with AB dividing earlier and transversely and P1 dividing later and
longitudinally. The GLP-1 transmembrane receptor, related to
Drosophila Notch, is first detectable late in the 2-cell stage at the
interface of P1 and AB (purple). The putative transcription factor
SKN-1 accumulates to high levels in the nucleus of P1 by late in the
2-cell stage (blue). Cytoplasmic MEX-3 (green) is a putative RNAbinding protein with KH domains. Dark green in AB indicates higher
levels relative to the light green in P1. (D) A 4-cell-stage embryo,
when a dorsal-ventral axis is established. The transcription factors
PAL-1 (red) and SKN-1 (blue) are present in the nuclei of P2 and
EMS. PAL-1 specifies the identity of two P2 descendants, C and D.
Low levels of MEX-3 persist in some posterior blastomeres at later
stages (not shown). (E) A 12-cell-stage embryo. Two ABa
granddaughters (shaded grey) respond to a signal from MS by
adopting fates that include the production of pharyngeal cells. By the
12-cell-stage SKN-1 is no longer detectable, but PAL-1 expression
persists. C produces epidermis and body wall muscle, MS produces
pharynx and body wall muscle, and E produces all of the intestine. P3
divides to produce D, a body wall muscle precursor, and P4, the
germline progenitor. See Sulston et al., 1983, for a complete
description of the embryonic lineage. See text for additional
references.
3818 B. Bowerman, M. K. Ingram and C. P. Hunter
mutant embryos, all pharyngeal cell production is skn-1dependent and none appears glp-1-dependent.
Pharyngeal cell production by P1 and AB in par
mutant embryos
To determine conclusively the origins of the pharyngeal muscle
made by par mutant embryos, we analyzed the abilities of
mutant P1 and AB blastomeres, ‘isolated’ by laser ablation (see
Materials and Methods), to produce pharynx. Based on the ap distribution of pharyngeal cells in terminally differentiated
mutant embryos (Fig. 2), it appears that in par-1, par-2 and
par-3 mutants, both P1 and AB can produce pharyngeal cells.
However, of those par-4 mutant embryos that make pharyngeal
cells, they do so only in the posterior part of the embryo, presumably from P1. Using laser ablation experiments, we found
that, as reported previously for par-1 mutant embryos
(Bowerman et al., 1993), both P1 and AB from par-3 mutant
embryos almost always produced large numbers of pharyngeal
muscle cells, and they did so independent of glp-1 function
(Table 1). In contrast to par-1 and par-3, isolated AB blastomeres from par-2 mutant embryos often produce no or few
pharyngeal muscle cells (Table 1), consistent with the observation that par-2 embryos produce glp-1-dependent pharyngeal
cells anteriorly (Fig. 2G,H). To test more conclusively if AB
descendants in par-2 mutant embryos produce glp-1-dependent
pharyngeal cells, we killed the P1 descendants in par-2 mutant
embryos after the time at which pharyngeal induction occurs
in wild-type embryos (Mango et al., 1994; Hutter and
Schnabel, 1995). We found that, when P1 descendants were
killed at these later stages, AB descendants always produced
large numbers of pharyngeal muscles cells (Table 1). Furthermore, when P1 descendants in par-2; glp-1 double mutant
embryos were killed at these later stages, AB descendants did
not make pharynx (Table 1). We conclude that production of
pharynx by AB descendants in par-2 mutant embryos requires
glp-1-dependent cell interactions. Finally, both in par-4 single
mutant embryos and in glp-1; par-4 double mutant embryos,
about 50% of isolated P1 blastomeres produced large numbers
of pharyngeal cells, while AB descendants never produced
pharynx (Table 1). This finding correlates well with the observation that 50% of intact par-4 mutant embryos produce pharyngeal cells posteriorly (Fig. 2G).
Pharyngeal cell production by P1 and AB daughters
in par mutant embryos
We further analyzed the patterning of pharynx by examining
the abilities of individual blastomeres in 4-cell-stage par
mutant embryos to produce pharyngeal muscle. In wild-type
embryos only one P1 daughter and one AB daughter produce
pharynx (Sulston et al., 1983). However, in par-1 mutant
embryos, both P1 and both AB daughters usually produce pharyngeal cells (Bowerman et al., 1993). Because the cleavage
axes of P1 and AB in par-2 and par-4 are highly variable, it
was not possible to reproducibly identify individual 4-cell-
Fig. 2. glp-1 and skn-1 function and the production of pharyngeal muscle cells in par-1, par-2, par-3 and par-4 mutant embryos. Fluorescence
micrographs of terminally differentiated wild-type (A) and mutant (B-O) embryos stained with monoclonal antibody 9.2.1 to visualize
pharyngeal muscle cells (Miller et al., 1983). Embryos are oriented with anterior to the left. Genotypes are indicated by labels to left and at top.
(A) Wild-type embryo near hatching with both P1- and AB-derived pharynx. (B) glp-1(e2142ts) embryo with only P1-derived pharynx. (C) skn1(zu67) embryo with no pharynx (D, E) par-1(e2012) and glp-1(e2142ts); par-1(e2012) embryos made indistinguishable amounts of
pharyngeal muscle. (F) skn1(zu67); par-1(e2012) embryo with no pharyngeal muscle [see Bowerman et al., 1993, for quantitation of par-1 and
glp-1; par-1 and skn-1; par-1 pharyngeal muscle phenotypes]. (G) par-2(lw32) embryos made large clumps of pharyngeal muscle, but (H) par2(lw32) glp-1(e2142ts) embryos made much less pharyngeal muscle, often in two small clumps. (I) par-2(lw32); skn-1(zu67) embryo with no
pharyngeal muscle: 111/128 par-2; skn-1 embryos made no pharyngeal muscle and 17/128 made only 2-8 pharyngeal muscle cells. (J,K) par3(it71) and par-3(it71) glp-1(e2142ts) embryos made indistinguishable amounts of pharyngeal muscle. (L) par-3(it71); skn-1(zu67) embryo
with no pharynx: 170/189 par-3; skn-1 embryos made no pharyngeal muscle and 19/189 made 2-16 pharyngeal muscle cells. Similar results
were obtained using par-3(it62); skn-1(zu67) embryos (data not shown). (M,N) par-4(it47ts) and glp-1(e2142ts); par-4(it47ts) mutant embryos
with large clumps of pharyngeal muscle cells located posteriorly. 234/424 par-4 embryos (55%) made pharyngeal muscle cells (10 to >50 cells/
embryo; 30 to 40 cells in most embryos). 190/424 (45%) made no pharyngeal muscle cells. (O) skn-1(zu67); par-4(it47) embryo with no
pharyngeal muscle cells. 372/395 skn-1; par-4 embryos made no pharyngeal cells; 23/395 made from 2 to 9 pharyngeal muscle cells. See
Materials and Methods for a description of the strains and alleles used and for fixation and antibody staining procedures.
Patterning in par mutants 3819
Table 1. Production of pharyngeal muscle cells and
intestinal cells by AB and P1
Pharynx
Wild-type
glp-1(e2142)*
par-2(lw32)
par-2(lw32)†
par-2(lw32) glp-1(e2142)*
par-3(it71)
par-3(e2059)
par-3(it71) glp-1(e2142)*
par-4(it57)
glp-1(e2142); par-4(it57)
Intestine
AB
P1
AB
P1
0/8
0/7
5/12
8/8
1/12
11/11
17/19
9/10
0/8
0/6
10/10
8/8
21/23
−
8/9
5/5
12/12
5/5
9/15
5/9
0/14
0/9
0/14
0/10
0/15
4/51
4/19
2/14
0/8
0/9
11/11
12/12
9/19
−
3/12
2/40
8/12
3/8
0/19
0/11
Results are recorded as no. of partial embryos that made intestinal cells or
pharyngeal cells/no. of partial embryos analyzed. 2-cell-stage embryos were
collected from wild-type or homozygous mutant mothers, and P1 or AB killed
with a laser microbeam. Partial embryos were allowed to develop overnight at
room temperature. Intestinal cells were scored by using polarizing optics to
detect the presence or abence of birefringent gut granules. The partial
embryos were then fixed and stained with monoclonal antibody 9.2.1 to score
the presence or absence of pharyngeal muscle cells (Miller et al., 1983). In all
antibody staining experiments, unablated embryos that made pharyngeal
muscle cells were used as positive controls. In par mutant embryos, P1 and
AB were defined by their position relative to the polar bodies, present at the
anterior end in 90% of the embryos but at the posterior end in 10%. Therefore
P1 and AB were accurately defined in only about 90% of the experiments
involving par embryos. As expected, we found that terminally differentiated
par-2; skn-1 and par-3; skn-1 double mutant embryos almost always fail to
produce intestinal cells (data not shown).
*All glp-1 and par glp-1 and par-4 embryos were obtained from mothers
raised at 25°C for at least 2 hours before collection. All other embryos were
obtained from mothers raised at room temperature.
†In these experiments, P1 descendants were killed at the 12-cell stage (see
text).
stage blastomeres. As the daughters cannot be distinguished,
we report the number of randomly isolated P1 daughters that
made pharyngeal cells/the number of embryos analyzed in par2, par-3 and par-4 mutant embryos. In par-2 mutants, 8/9
isolated P1 daughters made pharyngeal cells and 6/6 AB
daughters made pharyngeal cells, with the latter ablations done
at the 12-cell stage. In par-3 mutants, 3/3 isolated anterior P1
daughters, 4/4 isolated posterior P1 daughters, 5/6 isolated
anterior AB daughters and 3/3 isolated posterior AB daughters
made pharygneal muscle. In par-4 mutants, 5/9 isolated P1
daughters and 0/6 isolated AB daughters made pharyngeal
muscle. In all cases scored positive, 5-20 pharngeal muscle
cells were present. We conclude that the mechanisms normally
limiting pharyngeal cell production to only one P1 and one AB
daughter are not functional in par mutant embryos.
SKN-1 distribution in par mutant embryos
As the final step in our analysis of pharyngeal cell fate patterning in par mutant embryos, we examined the spatial and
temporal regulation of SKN-1 expression. In wild-type
embryos, SKN-1 is present at high levels only in P1 and its
descendants from late in the 2-cell stage until the 8-cell stage.
Peak levels of SKN-1 are detected at the 4-cell stage in EMS
and P2; little or no SKN-1 is detectable in ABa and ABp
(Bowerman et al., 1993; see Fig. 3A). As shown previously,
SKN-1 is present at roughly equal levels in all 4-cell-stage par1 blastomeres (Bowerman et al., 1993; Guo and Kemphues,
1995; see Fig. 3B). To determine if mislocalization of SKN-1
is a general property of par mutants, we examined par-2, par3 and par-4 mutant embryos. We found that, like par-1 mutants,
most par-3 mutants have roughly equal SKN-1 levels in all 4cell-stage blastomeres (Fig. 3B,D). However, SKN-1 localization appears normal in par-2 and par-4 mutant embryos (Fig.
3C,E). In contrast to the defects in spatial regulation, temporal
regulation of SKN-1 expression was not affected by mutations
in any of the par genes (see Fig. 3 legend). The ubiquitous
expression of SKN-1 in par-1 and par-3 mutant embryos correlates with the ability of both P1 and AB to produce skn-1dependent, glp-1-independent pharyngeal cells. Localization of
SKN-1 expression to P1 descendants in par-2 and par-4 mutant
embryos correlates with the ability of only P1 but not AB
descendants to produce glp-1-independent pharynx in par-2 and
par-4 mutants. The absence of ABa-derived pharyngeal cells in
par-4 mutant embryos could be due to a failure in signaling by
P1 descendants, or to a defective response by AB descendants.
Intestinal cell production by P1 and AB in par mutant
embryos
We next examined par mutant embryos with respect to another
a-p asymmetry, the production of intestinal cells by only P1 and
not AB (Sulston et al., 1983). Like the production of pharynx,
the production of intestine by P1 requires skn-1 function
(Bowerman et al., 1992). Although skn-1 is required in par
mutant embryos to specify the excess pharynx that they make,
most par mutant embryos fail to produce any intestinal cells
(Kemphues et al., 1988). Indeed, par-1 and par-4 mutant
embryos almost never make intestine. However, the fraction of
par-2 and par-3 mutant embryos that make intestinal cells is
sufficiently high to to determine the abilities of P1 and AB to
produce intestine (Table 1). We found that, in par-2 mutants,
only P1 and not AB is capable of producing intestinal cells. By
contrast, in par-3 mutant embryos, P1 and AB both produced
intestinal cells at a similar frequency. The production of intestinal cells by only P1 in par-2 mutant embryos, and by both P1
and AB in par-3 mutant embryos is consistent with the normal
and abnormal expression pattern of SKN-1 in, respectively,
par-2 and par-3 mutant embryos.
Patterning of body wall muscle in par mutant
embryos
The final asymmetry that we analyzed is in the different abilities
of P1 and AB to produce body wall muscle. In wild-type
embryos, P1 produces 80 of the 81 body wall muscle cells made
during embryogenesis in C. elegans (Sulston et al., 1983).
Although the production of body wall muscle by some P1
descendants is influenced by cell signals (Schnabel, 1994, 1995),
P1 nevertheless produces many body wall muscle cells after
removing or killing AB, while AB fails to produce body wall
muscle after removal of P1 (Priess and Thomson, 1987; Draper
et al., 1996). To further define the consequences of mutationally
inactivating the par genes, we examined the abilities of P1 and
AB to produce body wall muscle in par mutant embryos.
As with pharyngeal muscle production, the different abilities
of P1 and AB in par mutant embryos to produce body wall
muscle is apparent when differentiated intact mutant embryos
are fixed and stained with antibodies specific for a body
wall muscle myosin. par-1 (Fig. 4C) and par-3 (Fig. 4G)
mutants both produce body wall muscle throughout the
3820 B. Bowerman, M. K. Ingram and C. P. Hunter
embryo, suggesting that both P1 and AB make muscle, an
observation confirmed by laser ablation experiments (Table 2).
In par-2 mutant embryos, body wall muscle cells are made
only in the posterior part of the embryo (Fig. 4E). Laser
ablation experiments confirmed the impression from intact
embryos that only P1 and not AB produces body wall muscle
in par-2 embryos (Table 2). The body wall muscle phenotype
of par-4 mutant embryos is, like the pharyngeal phenotype,
more variable. par-4 mutant embryos produce posteriorly
localized body wall muscle cells in about 50% of the embryos;
the remaining embryos do not produce body wall muscle
(Table 2, Fig. 4 legend). To determine if the production of body
wall muscle and pharynx are coupled in par-4 mutant embryos,
we double-stained differentiated par-4 mutants with antibodies
both to body wall muscle and to pharyngeal muscle. We found
that about 43% of par-4 mutant embryos produce both pharyngeal and body wall muscles posteriorly (48/112). Surprisingly, though, some embryos produce only pharyngeal muscle
cells (3/112) and some produce only body wall muscle
(17/112). Thus, in some par-4 mutant embryos, the specification of pharyngeal muscle and body wall muscle appear
unlinked. Uncoupling of skn-1-dependent specification of
pharynx from muscle has been noted previously: Schnabel
(1994) showed that MS requires glp-1 function to produce
body wall muscle but not to produce pharyngeal muscle.
PAL-1 distribution in par mutant embryos
To further investigate the body wall muscle phenotype of par
mutant embryos, we analyzed the distribution of the homeodomain protein PAL-1. In wild-type embryos, body wall
muscle production is specified by both PAL-1 and SKN-1
(Mello et al., 1992; Bowerman et al., 1993; Hunter and
Kenyon, 1996), with PAL-1 detectable at the 4-cell stage in
only EMS and P2 (Hunter and Kenyon, 1996; see Fig. 5). PAL1 continues to be expressed in the descendants of both P1
daughters through the 28-cell stage (Table 3), although
expression appears stronger in P2 than in EMS descendants
(Hunter and Kenyon, 1996). Previous studies have shown that
PAL-1 is not expressed in par-1 mutant embryos (Hunter and
Kenyon, 1996). To determine if loss of PAL-1 expression is a
general property of par mutants, we examined par-2, par-3 and
par-4 embryos (Fig. 5; Table 3). In par-2 mutants, PAL-1 is
expressed in a wild-type pattern, although the expression level
appears to be reduced compared to wild-type (Fig. 5H). The
distribution of PAL-1 in par-3 mutant embryos was variable.
In about half the 4-cell to 32-cell par-3 mutant embryos, PAL1 was detected in a normal posterior-localized pattern (Fig. 5I).
Embryos that did not express PAL-1 in a wild-type pattern
either failed to produce any detectable PAL-1 or expressed
PAL-1 in all cells. The levels of PAL-1 observed in anterior
blastomeres of par-3 mutant embryos were often lower than
Fig. 3. SKN-1 distribution in par-1, par-2, par-3 and par-4 mutant
embryos. Fluorescence micrographs showing fixed 4-cell-stage
wild-type and par mutant embryos stained for SKN-1 protein (left
column) and with DAPI to visualize DNA in nuclei (right
column). Embryos are oriented with anterior to the left as
determined by the position of DAPI-stained polar bodies (visible
in I and J; out of the focal plane in the other DAPI images).
(A,F) Wild-type embryo with high levels of SKN-1 in P 2 and
EMS. Lower levels are just detectable in ABa and ABp. Note that
the chromosomes are more highly condensed in ABa and ABp
than in P2 and EMS. In DAPI-stained par mutant embryos (G-I),
chromosome condensation appears equivalent as all four cells
divide synchronously. (B,G) par-1(2012) mutant embryo. In 22/24
4-cell-stage par-1 embryos, equal levels of staining were detected
in all blastomeres; in 2/24 embryos, slightly lower levels of SKN1 were detected in the two more anterior blastomeres. Similar
results were obtained with par-1(b274) embryos (data not shown).
(C,H) par-2(lw32) mutant embryo. In 10/13 4-cell-stage par-2
embryos, SKN-1 was detectable in the two most posterior
blastomeres and undetectable or barely detectable in the two most
anterior blastomeres; in 3/13 slightly lower levels of SKN-1 were
detected in the two more anterior blastomeres. Similar results were
obtained using par-2(it5ts) embryos (data not shown). (D,I) par3(it71) mutant embryo. In 8/14 4-cell-stage par-3 mutant embryos,
SKN-1 was evenly distributed; in 3/14, one 4-cell-stage
blastomere stained less brightly than the other three; in 2/14, the
two anterior blastomeres stained less brightly; in 1/14, the two
posterior blastomeres stained less brightly. In all cases, SKN-1
was distributed more evenly than in wild-type. Similar results
were obtained using par-3(it62) embryos (data not shown).
(E,J) par-4(it57) mutant embryo. In 9/11 4-cell-stage par-4 mutant
embryos, high levels of SKN-1 were present in the two smaller
posterior blastomeres with no or little SKN-1 detectable in the two
larger anterior blastomeres; in 2/11 nearly equal levels of SKN-1
were detected in all four blastomeres. As in wild-type embryos, SKN-1 was detectable at low levels in all 2-cell-stage and 4-cell-stage par
mutant embryos examined [n=18, 8, 16, and 14 for par-1, par-2, par-3 and par-4 mutant embryos, respectively]. SKN-1 was detectable in
some but not all 8-cell-stage mutant embryos: 4/18 par-1, 2/9 par-2, 3/14 par-3 and 2/7 par-4 embryos had detectable staining,
frequencies similar to wild-type (Bowerman et al., 1993). In all embryos, SKN-1 was not detectable by the 16-cell stage (data not shown).
Patterning in par mutants 3821
Table 2. Production of body wall muscle cells by P1 and
AB
P1
AB
9/9
0/11*
11/11
0/7*
6/6
0/9*
par-2(lw32)‡
par-2(lw32) glp-1(e2142)†
par-2(lw32); skn-1(zu67)
21/23
9/10
11/12
1/20
1/12
1/10
par-3(it71)
par-3(e2059)
par-3(it71) glp-1(e2142)†
par-3(it71); skn-1(zu67)§
13/13
4/4
4/5
22/25
15/16
13/16
9/10
2/19
7/7
9/9
10/15
6/11
6/16
0/8
0/9
0/12
Wild-type
skn-1(zu67)
glp-1(e2142)†
par-2(lw32) par-3(it71)
par-4(it57)
glp-1(e2142); par-4(it57)
skn-1(zu67); par-4(it57)¶
Results are recorded as no. of partial embryos that made muscle cells/no. of
of partial embryos analyzed. 2-cell-stage embryos were collected from wildtype and homozygous mutant mothers, and P1 or AB were killed with a laser
microbeam. Partial embryos were allowed to develop for 8-10 hours at room
temperature, fixed and stained with the monoclonal antibody 5.6 to detect the
presence or absence of body wall muscle cells (Miller et al., 1983). In all
experiments, unablated embryos that made muscle cells were used as positive
controls. In about 90% of the partial par mutant embryos of all genotypes that
made muscle cells, 18->50 cells stained positively. In about 10% of the partial
par embryos of all genotypes that were scored as producing muscle cells,
only 2-14 muscle cells stained positively.
*AB usually produced 3-4 muscle cells in these experiments, as ABp
descendants produce one body wall muscle cell and three muscle cells
associated with the rectum in response to a signal from P2 at the 4-cell stage
that is difficult to eliminate by laser ablation. par mutant embryos of all
genotypes that were scored as not producing muscle did not make any muscle
cells.
†All glp-1 and par glp-1 embryos were obtained from mothers raised at
25°C for at least 2 hours before collection. All other embryos were obtained
from mothers raised at room temperature.
‡Similar results were obtained using par-2(it5ts) embryos obtained from
mothers raised at 25°C (data not show).
§Similar results were obtained using par-3(it62); skn-1(zu67) embryos
(data not shown). For additional details, see Materials and Methods.
¶skn-1; par-4 mutant embryos that made muscle made 8-22 muscle cells,
fewer than than the number usually made by P1 in par-4 mutant embryos
(18->50).
observed in posterior blastomeres (Fig. 5J), but in some cases
the levels were equal (Fig. 5K). Finally, we found that similar
to par-1, early stage par-4 mutant embryos do not make
detectable levels of PAL-1 (Fig. 5L).
skn-1 and pal-1 activities and the specification of
body wall muscle in par mutant embryos
In wild-type embryos, pal-1 is required for somatic P2 descendants to make body wall muscle, and skn-1 is required for the
normal specification of body wall muscle in EMS descendants
(Mello et al., 1992; Bowerman et al., 1993; Draper et al., 1996;
Hunter and Kenyon, 1996). Since null mutations in pal-1 are
zygotic lethals (L. Edgar and W. Wood, personal communication), we inhibited maternal pal-1 function by microinjecting
pal-1 antisense RNA into the syncitial ovary of pal-1(+) hermaphrodites (Hunter and Kenyon, 1996). Depleting pal-1
Fig. 4. skn-1 function and body wall muscle cell production in par1, par-2, par-3 and par-4 mutant embryos. Fluorescence
micrographs of differentiated wild-type (A) and mutant (B-J)
embryos stained with monoclonal antibody 5.6 to detect body wall
muscle cells (Miller et al., 1983). (A) Wild-type embryo showing
two of four longitudinal quadrants of body wall muscle. (B) skn1(zu67) embryo with body wall muscle in posterior part of embryo.
(C) par-1(e2012) embryo with body wall muscle throughout
embryo. (D) par-1(e2012); skn-1(zu67) with no body wall muscle
[see Bowerman et al., 1993, for quantitation]. (E) par-2(lw32)
embryo with body wall muscle in posterior part of embryo. (F) par2(lw32); skn-1(zu67) embryo with body wall muscle in posterior
part of embryo. (G) par-3(it71) embryo with body wall muscle
througout embryo. (H) par-3(it71); skn-1(zu67) embryo with body
wall muscle in posterior part of embryo [see Table 2 for
quantitation of body wall muscle production by par-2, par-3, par2; skn-1 and par-3; skn-1 mutant embryos]. (I) par-4(it47ts)
embryo with body wall muscle in posterior part of embryo. 144/283
par-4 embryos (51%) made body wall muscle cells (20-50
cells/embryo). 139/283 (49%) made no body wall muscle cells.
(J) skn-1(zu67); par-4(it47ts) embryo with body wall muscle in
posterior part of embryo. 228/423 skn-1; par-4 embryos (54%)
made body wall muscle cells (15-48 cells/embryo); 195/423 made
no body wall muscle.
function by injection of pal-1 antisense RNA into skn-1 mutant
hermaphrodites produces embryos that entirely lack body wall
muscle cells, suggesting that skn-1 and pal-1 define a minimum
set of body wall muscle specification activities (Hunter and
Kenyon, 1996; but see below).
To determine the contributions of skn-1 and pal-1 to the
3822 B. Bowerman, M. K. Ingram and C. P. Hunter
Table 3. PAL-1 staining in par mutant embryos
Posterior
nuclei only
All nuclei
stain
No
staining
WT
2-cell
4-cell
8-cell
12-32-cell
−
14
14
19
−
−
−
−
17
−
−
−
par-2
2-cell
4-cell
8-cell
12-32-cell
−
8
4
4
−
−
−
−
5
2
−
−
par-3
2-cell
4-cell
8-cell
12-32-cell
−
11
18
10
2*
7
5
2
30
11
10
5
par-4†
2-cell
4-cell
8-cell
12-32-cell
−
−
−
−
1
1
2
1
12
17
14
10
Results are recorded as the number of embryos detected showing the
staining pattern indicated at the top using antibodies specific for PAL-1. The
genotype and stage of the embryos analyzed are indicated at the left. Alleles
used are same as those in Fig. 5. For staining procedure, see the Materials and
Methods.
*The staining in the 2-cell embryos was very weak compared to staining in
the 4-cell and older embryos.
†The staining in ‘all nuclei’ was barely detectable.
Fig. 5. PAL-1 distribution in par-2, par-3 and par-4 mutant embryos.
Fluorescence micrographs showing fixed 4-cell-stage wild-type and
par mutant embryos stained with antibodies specific for PAL-1 (right
column) and with DAPI (left column). See Table 3 for quantitation.
(A,G) Wild-type embryo with high levels of PAL-1 in P2 and EMS.
(B,H) par-2(lw32) 4-cell-stage embryos usually have PAL-1 present
only in two posterior blastomeres (H); one anterior nucleus is out of
the focal plane (B). (C-E, I-K) par-3(it71) mutant embryos. PAL-1 is
present only in the posterior blastomeres of some 4-cell-stage
embryos (I), at higher levels posteriorly than anteriorly (J), and
sometimes at equal levels in all blastomeres, as in an 8-cell-stage
embryo (K). PAL-1 is not detectable in many par-3 mutant embryos
(see Table 3). (F,L) par-4(it57ts) 4-cell-stage mutant embryo with no
detectable PAL-1 (L).
specification of body wall muscle in par mutant embryos, we
removed or inhibited their activities in each par mutant. In par1 embryos both P1 and AB produce body wall muscle, SKN-1
is detected in all early blastomeres, and PAL-1 is not detectable
(Bowerman et al., 1993; Hunter and Kenyon, 1996). In par-1;
skn-1 double mutant embryos, body wall muscle cells are not
made (Bowerman et al., 1993; Draper et al., 1996; see Fig. 4D).
Thus skn-1 activity is necessary for all body wall muscle production by both P1 and AB in par-1 mutant embryos.
In par-2 mutant embryos, only P1 produces body wall
muscle (Table 2), and both SKN-1 and PAL-1 are localized
normally to posterior blastomeres (Figs 3, 5; Table 3). In par2; skn-1 double mutants, the posterior half of the embryo still
produces body wall muscle (Fig. 4F), and laser ablation experiments confirm that P1 makes muscle (Table 3). Similarly,
depleting pal-1 function in par-2 mutants by injection of pal1 antisense RNA results in embryos that still produce posteriorly localized body wall muscle (data not shown). These results
suggest that, in par-2 mutant embryos, either or both SKN-1
and PAL-1 can specify body wall muscle.
In par-3 mutant embryos, both P1 and AB produce body wall
muscle cells, and both SKN-1 and PAL-1 can be mis-localized
to anterior blastomeres (Figs 3D, 5D, 5E; Table 3). However,
in par-3; skn-1 double mutants, body wall muscle is always
made by P1 descendants even though PAL-1 is not detectable
in 30% of par-3 mutant embryos. These data suggest that either
undetectable amounts of PAL-1 can function to specify body
wall muscle production, or that additional body wall muscle
factors are active in par-3 mutant embryos. Injection of pal-1
antisense RNA into par-3; skn-1 double mutant hermaphrodites did significantly reduce or eliminate body wall muscle
production (18/31 embryo produced four or fewer muscle
staining cells), indicating that the posteriorly localized PAL-1
can specify body wall muscle in par-3 mutant embryos.
In par-4 mutants, only P1 produces body wall muscle, and
it does so in only 50% of the embryos (Fig. 4; Table 2). PAL-
Patterning in par mutants 3823
1 is not expressed in par-4 mutants and skn-1 is posteriorly
localized (Figs 3, 5). Surprisingly, 50% of par-4; skn-1 double
mutants still make posteriorly localized body wall muscle
(Table 2). Since PAL-1 is not expressed in par-4 mutant
embryos, there may be a SKN-1/PAL-1-independent activity
that can specify body wall muscle production in par-4 mutant
embryos (see Discussion).
MEX-3 distribution in par mutant embryos
MEX-3 is a putative RNA-binding protein with KH domains that
is required to repress pal-1 translation in oocytes and early
embryos (Draper et al., 1996). MEX-3 is distributed throughout
oocytes but at the 4-cell stage is present cytoplasmically at higher
levels in anterior than in posterior blastomeres (Draper et al.,
1996; see Fig. 6A). Previous studies have shown that, in mex-3
mutant embryos, PAL-1 is strongly expressed in all blastomeres,
and all blastomeres produce body wall muscle (Draper et al.,
1996; Hunter and Kenyon, 1996). Furthermore, it has been
proposed that the higher levels of MEX-3 in anterior blastomeres
at the 4-cell stage might limit pal-1 translation to posterior blas-
tomeres. Consistent with this hypothesis are the observations that
MEX-3 is present at high levels in all blastomeres and PAL-1 is
not detectable in par-1 mutant embryos. Moreover, removing
mex-3 activity from par-1 embryos relieves the translational
repression and results in PAL-1 being produced at high levels in
all blastomeres (C. P. H., unpublished data). Similarly a mex-3;
skn-1; par-1 triple mutant embryo produces large numbers of
body wall muscle cells from both P1 and AB (Draper et al., 1996).
Thus, low levels of MEX-3 correlate with PAL-1 expression in
wild-type embryos and in mex-3 and par-1 mutants.
To further test the relationship between MEX-3 and PAL-1,
we stained par-2, par-3 and par-4 mutant embryos with a monoclonal antibody specific for MEX-3 (Fig. 6; Table 4). Similar
to wild-type embryos, in par-2 mutants PAL-1 is detected in both
P1 daughters. Consistent with the translational repression model,
we found that the MEX-3 distribution also appeared wild-type,
with higher levels at the 4-cell stage in AB daughters than in P1
daughters. In par-3 mutants, PAL-1 is present at detectable levels
in all blastomeres in some 4-cell and 8-cell-stage embryos, and
only posteriorly in others (Fig. 5; Table 3). Surprisingly, we
found that MEX-3 was present at high levels in the cytoplasm
of all 4-cell-stage blastomeres in all par-3 mutant embryos
examined (Fig. 6C). Thus, in par-3 mutant embryos, MEX-3
levels do not correlate with the pattern of PAL-1 expression.
Finally, par-4 mutant embryos resemble par-1 mutants: no PAL1 expression was detected in par-4 mutant embryos (Fig. 5L)
and MEX-3 was present cytoplasmically at high levels in all 4cell-stage par-4 mutant embryos (Fig. 6D). Like mex-3; par-1
double mutant embryos, mex-3; par-4 double mutant embryos
express PAL-1 in all blastomeres (C. P. H., unpublished data).
Thus the ubiquitously distributed MEX-3 appears to repress all
PAL-1 expression in both par-1 and par-4 mutants. In summary,
Table 4. MEX-3 staining in par mutant embryos
Fig. 6. MEX-3 distribution in par-2, par-3 and par-4 mutant
embryos. Fluorescence micrographs showing fixed 4-cell-stage wildtype and par mutant embryos stained with antibodies specific for
MEX-3 protein (left column) and with DAPI (right column). For
quantitation of results, see Table 4. (A,E) Wild-type 4-cell-stage
embryo with high levels of MEX-3 in ABa and ABp (A). MEX-3 is
also a P-granule component (Draper et al., 1996) as indicated by the
punctate staining in P2 (A). (B,F) par-2(lw32) 4-cell-stage embryo
with MEX-3 at higher levels in anterior than in posterior blastomeres
(B). (C,G) par-3(it71) 4-cell-stage embryo with MEX-3 at high
levels in all blastomeres (C). MEX-3 is also detectable in P-granules
in P2 and EMS; some of these are visible in the par-2 embryo (B).
(D,H) par-4(it57ts) 4-cell-stage embryo with MEX-3 at high levels in
all blastomeres (D).
High levels
anteriorly
Equal in
all cells
No
staining
WT
2-cell
4-cell
8-cell
16-cell
12
32
3
−
14
7
−
−
1
−
−
7
par-2
2-cell
4-cell
8-cell
16-cell
9
25
4
−
14
8
3
−
1
1
−
6
par-3
2-cell
4-cell
8-cell
12-20 cell
−
−
−
−
14
21
8
−
−
−
−
5
par-4
2-cell
4-cell
8-cell
16-cell
2
1
−
−
30
34
6
−
1
−
−
12
Results are recorded as the number of embryos detected showing the
staining pattern indicated at the top using antibodies specific for MEX-3. The
genotype and stage of the embryos analyzed are indicated at the left. Alleles
used are same as those in Fig. 6.
3824 B. Bowerman, M. K. Ingram and C. P. Hunter
although the MEX-3 and PAL-1 expression patterns are complementary in wild-type, par-1, par-2 and par-4 mutant
embryos, they are not so correlated in par-3 mutant embryos.
DISCUSSION
The par genes sometimes act independently of each
other to polarize and pattern early C. elegans
embryos
We have shown that the phenotypes of par-1, par-2, par-3 and
par-4 mutant embryos, with respect to the function of skn-1,
glp-1, pal-1 and mex-3, each are unique. We briefly summarize
each mutant and then discuss the implications of this diversity
in phenotype. (i) par-1 mutations, as shown previously, result
in ubiquitous expression of GLP-1, MEX-3 and SKN-1, and
cause a loss of PAL-1 expression. par-1 mutant embryos
produce skn-1-dependent, glp-1-independent pharynx and body
wall muscle from all 4-cell-stage blastomeres. (ii) par-2 mutant
embryos show normal localization of SKN-1, PAL-1 and MEX3 at the 4-cell stage. Both posterior blastomeres in 4-cell-stage
par-2 mutant embryos produce skn-1- and pal-1-dependent
body wall muscle and skn-1-dependent, glp-1-independent
pharynx. Both anterior blastomeres produce glp-1-dependent
pharynx. (iii) par-3: As in par-1 mutants, SKN-1 and MEX-3
are expressed ubiquitously in par-3 mutant embryos. But PAL1, instead of being absent, is expressed and sometimes mislocalized. All 4-cell-stage blastomeres produce skn-1dependent, glp-1-independent pharynx and body wall muscle.
(iv) par-4 mutant embryos are unusual in that about 50% of
them fail to produce any pharynx or body wall muscle, even
though SKN-1 is expressed normally. As in par-1 mutants,
MEX-3 is expressed ubiquitously and PAL-1 is absent.
Based on their lack of phenotypic similarity, we suggest that
the par genes do not regulate pattern formation by operating
in a single genetic pathway to control the polarity of early
embryonic cells. At the same time, some par phenotypes show
simple epistatic relationships: most notably, par-3 is epistatic
to par-2 with respect to mitotic spindle axis orientation in P1
and AB, with respect to SKN-1 distribution in P1 and AB
descendants, and with respect to the abilities of P1 and AB to
produce pharynx and body wall muscle (Cheng et al., 1995;
Tables 1 and 2). Thus some par gene functions probably
involve steps arranged in a linear pathway. However, for the
par genes as a group, we think it most likely that they function
as part of a complex network of interconnected pathways in
which some par gene products regulate aspects of blastomere
polarity and cell fate patterning independently of others.
Epistasis analysis of cell fate specification pathways in other
par double mutants may shed more light on this issue.
In addition to each par mutant exhibiting a unique phentoype,
we note that none of the four par genes that we analyzed are
required for all a-p asymmetries. par-1 mutants show the most
extensive loss of a-p asymmetry: (1) the first cleavage is equal;
(2) P-granules, SKN-1, GLP-1 and MEX-3 are present at equal
levels in anterior and posterior blastomeres, and (3) PAL-1 is
absent from all blastomeres. However, as in wild-type, the
mitotic spindle is oriented longitudinally in the posterior 2-cellstage blastomere and transversely in the anterior blastomere,
indicating that some a-p asymmetry remains in par-1 mutants.
par-3 mutant embryos, like par-1 mutants, show extensive
losses in a-p asymmetry: P1 and AB both divide longitudinally,
and SKN-1, MEX-3 and, to a lesser extent, GLP-1 are all mislocalized. However, PAL-1 protein and function appear to be
localized to posterior blastomeres in many par-3 mutant
embryos. par-4 mutants show a loss of asymmetry in MEX-3
and GLP-1 distribution and fail to express PAL-1 but, as in wild
type, the first cleavage is unequal and SKN-1 is present only in
posterior blastomeres. Surprisingly, par-2 mutant embryos
appear to show substantially less loss of a-p asymmetry.
Although the first cleavage in par-2 mutant embryos always
produces two equal-sized daughters, SKN-1, GLP-1, PAL-1 and
MEX-3 are localized normally in most par-2 mutant embryos
(our data; Crittenden et al., 1996), and P-granules are present
only in the posterior blastomere at the 2-cell stage (Kemphues
and Strome, 1997). The observation that no single par gene is
required for all a-p asymmetries is consistent with our conclusion that the par genes in some cases act independently of each
other to generate polarity in the early embryo.
Mislocalization of regulatory proteins and their
sufficiency to ectopically specify developmental
programs in par mutant embryos
The status of different cell fate determination pathways in par
mutant embryos also yields insight into the sufficiencies of
different regulators to promote or repress specific developmental
programs. We emphasize four such findings. (i) In par-3 mutant
embryos, MEX-3 levels do not correlate with PAL-1 expression
patterns (see below for discussion). (ii) In par-4 mutant embryos,
there may be a body wall muscle specification pathway independent of either skn-1 or pal-1 function: even though no PAL1 is detectable in par-4 mutant embryos, 50% of skn-1; par-4
double mutant embryos produce large numbers of body wall
muscle cells. Thus it may be significant that 50% of skn-1 mutant
embryos in which pal-1 function has been depleted by pal-1 RNA
injection produce body wall muscle (Hunter and Kenyon, 1996).
This latter result could be explained by incomplete depletion of
pal-1 activity, or it might also indicate that a SKN-1/PAL-1-independent activity can specify body wall muscle. (iii) Also in par4 mutants, we find that high levels of SKN-1 are not always sufficient to specify the production of pharyngeal cells and body wall
muscle cells, and we note that the induction of anterior pharyngeal cells never occurs. (iv) Because PAL-1 is expressed normally
in par-2 mutants, and PAR-1 is not cortically enriched in par-2
mutants (Guo and Kemphues, 1995), cortically localized PAR-1
is not required for PAL-1 expression in P1 descendants. Similarly,
P-granules are localized to the posterior blastomere in 2-cellstage par-2 mutant embryos, indicating the cortical PAR-1 is not
required for P-granule segregation during the first embryonic
cleavage (Kemphues and Strome, 1997).
PAL-1 translational control: repression,
derepression and localization
Previous studies have shown that spatial regulation of PAL-1
expression occurs at least in part translationally (Hunter and
Kenyon, 1996). One key regulator is the putative RNA-binding
protein MEX-3, which is expressed at high levels in oocytes but
becomes partially localized to anterior blastomeres in 4-cellstage embryos (Draper et al., 1996). The complementarity of
the MEX-3 and PAL-1 expression patterns suggest that the
levels of MEX-3 might regulate the pattern of pal-1 translation:
higher anterior MEX-3 levels may limit PAL-1 production to
posterior blastomeres. This hypothesis is supported by the
Patterning in par mutants 3825
MEX-3
GLP-1
SKN-1
PAL-1
WT
par-1(-)
par-2(-)
par-3(-)
par-4(-)
mex-1(-)
mex-3(-)
Fig. 7. Summary of MEX-3, GLP-1, SKN-1 and PAL-1 expression
patterns in par-1, par-2, par-3 and par-4 mutant embryos. The data
for MEX-3, SKN-1 and PAL-1 are from data presented here; the data
for GLP-1 are from Crittenden et al., 1996. GLP-1 is mislocalized in
37% of par-2 embryos and in 77% of par-3 embros; PAL-1 is
mislocalized in 24% of par-3 mutant embryos. In all other cases,
nearly all embryos show the pattern illustrated.
observations in par-1 mutant embryos that MEX-3 is present at
high levels in all 4-cell-stage blastomeres and that PAL-1 is not
detectable (Draper et al., 1996; Hunter and Kenyon, 1996).
Thus it is conceivable that, in wild-type embryos, par-1 negatively regulates mex-3 in posterior blastomeres, resulting in
lower levels of MEX-3 which permit translation of pal-1 mRNA
posteriorly. Our analysis of body wall muscle specification in
par-2 and par-4 mutant embryos support the original correlation between high levels of MEX-3 and translational repression
of pal-1 mRNA. In 4-cell-stage par-2 mutant embryos, as in
wild-type embryos, MEX-3 is present at higher levels in
anterior blastomeres and PAL-1 is detectable only in posterior
blastomeres. In par-4 mutant embryos, as in par-1 mutant
embryos, MEX-3 is present at high levels both anteriorly and
posteriorly and PAL-1 is not detectable. Alternatively, though,
par-1 could act independently of mex-3 to relieve repression of
pal-1 translation in posterior blastomeres, and the levels of
MEX-3 might be an indirect result of such de-repression.
The correlation between MEX-3 levels and PAL-1 expression
does not hold true in par-3 mutant embryos: MEX-3 is present
at high levels both anteriorly and posteriorly, and yet PAL-1 is
detectable often in posterior and occasionally in anterior blastomeres. Thus the levels of MEX-3 cannot account for the
pattern of pal-1 translation in par-3 mutant embryos. One interpretation of these results is that par-1 and par-4 are required for
an activity that relieves MEX-3-mediated repression of pal-1
translation without directly affecting MEX-3. This hypothesis
is supported by the observations that PAL-1 is not detectable in
par-1 or par-4 mutant embryos but is expressed in all blastomeres in mex-3; par-1 and mex-3; par-4 double mutant
embryos. Furthermore, although PAL-1 can be mislocalized in
par-3 mutant embryos, it is not detected at significant levels
until the 4-cell stage. This separation of temporal and spatial
regulation indicates that par-3 function may be required to
localize a par-1- and par-4-dependent pal-1 derepressing
activity to posterior blastomeres. Finally, it is important to bear
in mind that abnormal interactions might occur in par mutant
embryos due to the mislocalization of multiple regulatory
factors. If so, the loss of correlation between MEX-3 and PAL1 expression in par-3 mutant embryos could be misleading.
The mechanisms that localize different
developmental regulators are not tightly coupled in
early C. elegans embryos
Our results, summarized in Fig. 7, indicate that largely independent mechanisms control the localization of different regulatory proteins in the early C. elegans embryo. Even proteins
with similar expression patterns respond differently to loss of
par functions. For example, par-2 mutations uncouple the
anterior localization of GLP-1 and MEX-3, while par-1 and
par-4 mutations differently uncouple the posterior localization
of SKN-1 and PAL-1. The apparent lack of coupling of the
mechanisms that generate different a-p asymmetries in the
early C. elegans embryo is in substantial contrast to the largely
heirarchical localization of maternal gene products during the
generation of a-p asymmetry in the Drosophila melanogaster
oocyte (St. Johnston, 1993).
How directly or indirectly the par genes affect localization of
SKN-1, GLP-1, PAL-1 and MEX-3 expression remains to be
determined. The maternal RNAs for skn-1, glp-1 and pal-1 are
distributed evenly throughout early embryos (Evans et al., 1994;
Seydoux and Fire, 1994; Hunter and Kenyon, 1996). Presumably,
polarization of the early embryo by PAR proteins in response to
sperm entry localizes translational regulators, and perhaps regulators of protein or RNA stability, that in turn control the
expression of the regulatory proteins analyzed here. Indeed,
recent studies have shown that translational regulation requiring
3′UTR sequences from the corresponding mRNA appear sufficient to account for the localized expression of GLP-1 and PAL1 (Evans et al., 1994; Hunter and Kenyon, 1996). However, the
apparent uncoupling of the spatial regulation of different regulatory proteins in par mutant embryos suggest that the events
linking par gene functions to translational regulation may be
complex and indirect. While several genes that control specific
blastomere identities, and six par genes, have been identified in
C. elegans (Kemphues and Strome, 1997; Schnabel and Priess,
1997), the functional links between the events controlled by these
two different groups of maternal genes remain to be elucidated.
We are grateful to Bruce Draper and Jim Priess for providing MEX3 antibodies prior to publication. We thank the C. elegans stock center,
funded by the NIH, and Jim Priess and Ken Kemphues for some of the
strains used in these experiments; Sarah Crittenden and Judith Kimble,
Bruce Draper and Jim Priess, and Jennifer Watts, Diane Morton and Ken
Kemphues for sharing unpublished information; Michele Champagne,
Danielle Hamill, Bruce Howard and Elena Kouzminova for constructing double mutants; David Miller and Susan Strome for antibodies; and
Bruce Draper, Pierre Gönczy and Chris Shelton for helpful comments
and discussions. C. P. H. would like to thank and acknowledge Cynthia
Kenyon for her generosity in providing space and support (NIH
3826 B. Bowerman, M. K. Ingram and C. P. Hunter
GM37053) during some of the initial experiments of this investigation.
C. P. H. was supported by a grant from the American Cancer Society –
CA division. This worked was also supported by grants from the
American Cancer Society (DB-61) and the NIH (GM49869) to B. B.
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(Accepted 1 August 1997)