RESEARCH ARTICLE 1259
Development 134, 1259-1268 (2007) doi:10.1242/dev.02833
PAR-6 is required for junction formation but not apicobasal
polarization in C. elegans embryonic epithelial cells
Ronald Totong*, Annita Achilleos* and Jeremy Nance†
Epithelial cells perform important roles in the formation and function of organs and the genesis of many solid tumors. A
distinguishing feature of epithelial cells is their apicobasal polarity and the presence of apical junctions that link cells together. The
interacting proteins Par-6 (a PDZ and CRIB domain protein) and aPKC (an atypical protein kinase C) localize apically in fly and
mammalian epithelial cells and are important for apicobasal polarity and junction formation. Caenorhabditis elegans PAR-6 and
PKC-3/aPKC also localize apically in epithelial cells, but a role for these proteins in polarizing epithelial cells or forming junctions has
not been described. Here, we use a targeted protein degradation strategy to remove both maternal and zygotic PAR-6 from C.
elegans embryos before epithelial cells are born. We find that PKC-3 does not localize asymmetrically in epithelial cells lacking PAR6, apical junctions are fragmented, and epithelial cells lose adhesion with one another. Surprisingly, junction proteins still localize
apically, indicating that PAR-6 and asymmetric PKC-3 are not needed for epithelial cells to polarize. Thus, whereas the role of PAR-6
in junction formation appears to be widely conserved, PAR-6-independent mechanisms can be used to polarize epithelial cells.
INTRODUCTION
Epithelial cells are integral components of organs and perform
crucial roles in organ morphogenesis and function. As epithelial
cells form, they acquire an apicobasal polarity that distinguishes
their different membrane surfaces. Apical and basolateral surfaces
differ in part because of targeted protein secretion (Nelson and
Yeaman, 2001; Rodriguez-Boulan et al., 2004); junctions that form
near the apical-basolateral interface also limit mixing between the
two domains and promote adhesion by linking epithelial cells
together (Anderson et al., 2004). Epithelial polarity allows different
regions of the cell to develop specialized structures and functions,
and, based on studies of Drosophila tumor suppressor proteins, is
thought to inhibit cell proliferation and tumor formation (Humbert
et al., 2003). However, how epithelial cells first polarize and
assemble apical junctions is not well understood.
Many different cell types require the conserved scaffolding
protein PAR-6 to polarize (Macara, 2004). PAR-6, which contains
PB1, CRIB and PDZ domains that bind other polarity proteins such
as the atypical protein kinase C (aPKC) PKC-3 (Hung and
Kemphues, 1999; Joberty et al., 2000; Lin et al., 2000; Suzuki et al.,
2001), was first identified for its role in polarizing the C. elegans
zygote (Watts et al., 1996). The zygote is a highly polarized cell that
cleaves asymmetrically to produce anterior and posterior daughter
cells, which differ in size and developmental potential. PAR-6 and
PKC-3 become restricted to an anterior cortical domain of the
zygote and regulate the localization of developmental determinants
and proteins required for asymmetric cleavage (Colombo et al.,
2003; Gotta et al., 2003; Pellettieri and Seydoux, 2002). PAR-6 also
functions later to polarize early embryonic cells: in response to the
pattern of cell-cell contacts, PAR-6 becomes restricted to the outer
(contact-free) cortex of cells and regulates inner-outer asymmetries
Skirball Institute of Biomolecular Medicine and NYU School of Medicine, 540 First
Avenue, New York, NY 10016, USA.
*These authors contributed equally to this work
†
Author for correspondence (e-mail: [email protected])
Accepted 12 January 2007
in cell adhesion and cytoskeletal organization important for
gastrulation (Hung and Kemphues, 1999; Nance et al., 2003; Nance
and Priess, 2002).
PAR-6 homologues are required to polarize epithelial cells in
other organisms. Epithelial cells polarize either when clusters of
precursor cells receive polarity cues provided by their external
environment or when the membrane of the egg is divided into apical
and basolateral domains by cleavage or cellularization (Drubin and
Nelson, 1996; Muller, 2001). In Drosophila blastoderm epithelial
cells, which form by cellularization of syncytial embryonic nuclei,
Par-6 and aPKC localize to an apical domain adjacent to adherens
junctions (Petronczki and Knoblich, 2001; Wodarz et al., 2000).
aPKC requires Par-6 for its apical localization and regulates polarity
effectors via phosphorylation, suggesting that aPKC asymmetry is
critical for establishing polarity (Betschinger et al., 2003; Hutterer
et al., 2004; Plant et al., 2003; Yamanaka et al., 2003). When both
maternal and zygotic pools of Par-6 are removed, adherens junction
proteins and other apical proteins fail to localize, and epithelial cells
lose their laminar organization (Hutterer et al., 2004; Petronczki and
Knoblich, 2001). Par6 also regulates junction formation in cultured
mammalian epithelial cells, which polarize upon contact with
neighboring cells. Mammals contain four genes that encode different
Par6 isoforms (A-D) (Gao and Macara, 2004; Joberty et al., 2000).
Overexpressing full-length Par6B or truncated Par6A in canine
kidney epithelial cells inhibits tight junction assembly (Gao et al.,
2002; Gao and Macara, 2004; Joberty et al., 2000; Yamanaka et al.,
2001). In addition, Par6C in human kidney epithelial cells interacts
with TGF␤ receptors to mediate tight junction disassembly during
TGF␤-induced epithelial-to-mesenchymal transitions (Ozdamar et
al., 2005). However, because cells lacking all Par6 isoforms have not
been described, it is unclear whether mammalian Par6 regulates
aspects of epithelial polarization other than tight junction biogenesis.
C. elegans PAR-6 and PKC-3 localize asymmetrically within
several epithelial cell types, but a role for these proteins in epithelial
polarity or junction formation has not been described. In part, this
may be due to difficulties in removing both maternal and zygotic PAR
proteins from epithelial cells without also preventing polarization of
the zygote (Hung and Kemphues, 1999; Watts et al., 1996). C.
DEVELOPMENT
KEY WORDS: Polarity, Organogenesis, Epithelium, Cell junctions, PAR-6, C. elegans
1260 RESEARCH ARTICLE
par-6(M/Z) embryos
par-6(M/Z) embryos were obtained by crossing unc-101 +/+ par6(tm1425); him-8 males with unc-101 par-6(zu170); zuIs43 [pie1::gfp::par-6::zf1] hermaphrodites (P0 cross) and allowing the non-Unc F1
progeny to self-fertilize; 25% of F2 embryos are expected to be par-6(M/Z):
par-6(tm1425)/par-6(tm1425) homozygotes that express maternal PAR6ZF1GFP protein only during early embryonic stages. par-6(M/Z) embryos
were distinguished from their wild-type siblings in immunostaining
experiments by including PAR-6 or PKC-3 antibodies. Where indicated, the
par-6(zu222) mutation was used in place of par-6(zu170) or the zuIs44
transgene was used in place of zuIs43; all gave similar results.
par-6(M/Z) embryos expressing DLG-1GFP were obtained by replacing
unc-101 par-6(zu170); zuIs43 P0 hermaphrodites with unc-101 par6(zu170); zuIs43; xnIs17 [dlg-1::gfp].
par-6(M/Z) embryos depleted of DLG-1 or LET-413 were obtained by
injecting non-Unc F1 hermaphrodites from the P0 cross described above
with dsRNA from the dlg-1 or let-413 gene (see below).
hmr-1(zu248, RNAi) par-6(M/Z) embryos were obtained by replacing the
P0 male strain with hmr-1(zu248) + par-6(tm1425)/+ unc-101 +; him-8
males and placing non-Unc F1 hermaphrodites on hmr-1 RNAi feeding
plates (see below) to remove maternal HMR-1. hmr-1(zu248, RNAi) par6(M/Z) embryos were distinguished from siblings by co-staining with HMR1 and PKC-3 antibodies; only embryos with no detectable HMR-1 and
uniformly cytoplasmic PKC-3 in intestinal epithelial cells were scored. For
control hmr-1 embryos, hmr-1(zu248) hermaphrodites expressing an
extrachromosomal array containing hmr-1 (zuEx3) were placed on hmr-1
RNAi plates; only embryos lacking HMR-1 immunostaining in intestinal
epithelial cells were scored.
MATERIALS AND METHODS
RNAi
Strains
par-6 3⬘ UTR RNAi and hmr-1 RNAi was performed by the feeding method.
Base pairs 1-386 of the par-6 3⬘ UTR or bp 13,345-13,988 of the hmr-1a
gene (start codon=1) were amplified and cloned into RNAi feeding vector
pPD129.36 (Timmons and Fire, 1998). Feeding RNAi was performed as
described (Kamath et al., 2001), substituting ␤-lactose for IPTG (Gobel et
al., 2004). L4 hermaphrodites placed on feeding plates were incubated at
25°C and embryos laid after 36-48 hours were analyzed. In control
experiments using wild-type hermaphrodites, 241/245 par-6-UTR(RNAi)
embryos died and 15/16 embryos showed a Par first cleavage. All hmr1(RNAi) embryos died (103/103).
let-413 and dlg-1 RNAi was performed by the injection method. Coding
sequences from each gene were amplified from genomic DNA using T7promoter-tagged primers [let-413: bp 1014-1613; dlg-1: bp 3185-3675] and
dsRNA was produced by in vitro transcription (Nance and Priess, 2002).
Young adult hermaphrodites were injected with 1-3 ␮g/␮L dsRNA and
allowed to recover at 20°C; eggs laid 20-32 hours post-injection were
collected and immunostained. To monitor the extent of protein depletion
following RNAi, a subset of embryos in each experiment was stained for
LET-413 or DLG-1. Of the 30 let-413(RNAi) embryos 29 lacked intestinal
LET-413 staining. A total of 89/101 dlg-1(RNAi) embryos lacked intestinal
DLG-1 staining, 11/101 showed trace amounts of staining and 1/101 showed
normal levels of staining.
The following mutations and chromosomal rearrangements were utilized:
LGI: hmr-1(zu248) (Costa et al., 1998), unc-101(m1), par-6(zu170, zu222,
tm1425) (Watts et al., 1996); LGIII: unc-119(ed3); LGIV: him-8(e1489).
zuEx3 is an extrachromosomal array containing hmr-1(+) and rol-6(d)
(Costa et al., 1998). xnEx12 (this study) is an extrachromosomal array
containing par-6::gfp and unc-119(+) (Nance et al., 2003). The par6(tm1425) deletion was provided by the National Bioresource Project (S.
Mitani, Tokyo Women’s Medical University), outcrossed six times,
balanced, and sequenced. tm1425 removes bp 117 to 969 of the par-6
genomic sequence (start codon=1). par-6(tm1425) failed to complement
par-6(zu222) for the maternal-effect Par phenotype (12/12) and caused a
recessive larval lethal phenotype [2/135 eggs from par-6(tm1425)/+
hermaphrodite did not hatch, 40/135 (30%) progeny died as early larvae,
93/135 (69%) grew to fertile adults]. The larval lethal phenotype of par6(tm1425) homozygotes is rescued by the xnEx12 par-6::gfp transgenic
array: par-6(1425)/par-6(tm1425); xnEx12 hermaphrodites are viable,
fertile, and produce viable progeny [95/101 eggs developed to fertile adults].
Transformation
xnIs17 [dlg-1::gfp] was created by integrating a dlg-1::gfp
extrachromosomal array (Firestein and Rongo, 2001) using
trimethylpsoralen mutagenesis and UV irradiation (Clark and Chiu, 2003).
All other extrachromosomal and integrated transgenic lines were created by
biolistic transformation of unc-119 worms (Praitis et al., 2001).
Transgene construction
A transgene expressing PAR-6ZF1GFP driven by the maternal pie-1 promoter
was created by digesting the pie-1::gfp::actin plasmid pJH4.64 (Reese et al.,
2000) with SpeI to remove actin coding sequences, then replacing with par6 and zf1 coding sequences. pJH4.64 was also modified to eliminate an
internal NotI site. The complete par-6 cDNA (Hung and Kemphues, 1999)
was amplified by RT-PCR using an NheI-tagged forward primer and a SpeItagged reverse primer; digested RT-PCR product was inserted into the SpeI
site 3⬘ of gfp. The unc-119(+) gene from plasmid pJN254 (Nance et al.,
2003) was inserted into the NotI site. The zf1 coding sequence (codons 97132 from the pie-1 gene) (Reese et al., 2000) was amplified using SpeItagged primers and inserted into the SpeI site 3⬘ of par-6. Two transgenic
lines were obtained (zuIs43 and zuIs44).
Videomicroscopy
Three-dimensional timelapse Nomarski movies were acquired using a Zeiss
Imager equipped with a 63⫻ 1.4 NA or 40⫻ 1.3 NA objective, Nomarski
optics, an Axiocam MRM digital camera, and Axiovision software. Z-stacks
of sections spaced at 1 ␮m intervals were captured every 2-5 minutes.
Fluorescence timelapse movies of DLG-1GFP in hypodermal cells were
acquired using a Zeiss 510 LSM confocal microscope, 63⫻ 1.3 NA
objective, 488 nm laser at 9% strength and 2⫻ zoom. Laser intensity and
scan speed were adjusted so that imaged control embryos hatched.
Maximum intensity projections of several planes at 1 ␮m intervals were
compiled and optimized using ImageReady. For movies of par-6(M/Z)
embryos, mutant embryos and control sibling embryos were mounted
together. After acquiring movies, mutant embryos were distinguished as
those that arrested before the twofold stage and developed cell adhesion
defects; in all cases, arrested embryos expressing DLG-1GFP had fragmented
junctions.
DEVELOPMENT
elegans epithelial cells differentiate from clusters of precursor cells
that polarize during organogenesis. The first epithelial cells form
during the middle stages of embryogenesis and consist primarily of
epidermal cells, pharyngeal cells, and intestinal cells (see Fig. 1).
PAR-6 and PKC-3 localize to apical regions of pharyngeal and
intestinal epithelial cells (Bossinger et al., 2001; Leung et al., 1999;
McMahon et al., 2001). PAR-6 and PKC-3 are also found at apical
surfaces of reproductive tract epithelial cells, such as distal
spermathecal cells, that form during larval development (Aono et al.,
2004; Hurd and Kemphues, 2003). Reducing larval levels of the PDZ
domain protein PAR-3, which is required to localize PAR-6 and
PKC-3, disrupts the polarity of distal spermathecal cells (Aono et al.,
2004). However, because PAR-3 might have functions other than
regulating PAR-6 and PKC-3 localization, it is not known whether
PAR-6 and PKC-3 are needed to polarize spermathecal cells or any
other type of C. elegans epithelial cell.
Here we use a targeted protein degradation strategy to remove
both maternal and zygotic PAR-6 from C. elegans embryos before
epithelial cells form. We find that PKC-3 can no longer localize
asymmetrically in epithelial cells, and embryos arrest during
morphogenesis with severe defects in epithelial cell adhesion and
apical junction formation. Surprisingly however, epithelial cells are
still able to polarize. Thus PAR-6 has a widely conserved role in
regulating epithelial cell junctions but epithelial cells can utilize
PAR-6-independent mechanisms to polarize.
Development 134 (7)
C. elegans PAR-6 and junction formation
RESEARCH ARTICLE 1261
Immunostaining
RESULTS
PAR-6 expression and localization in embryonic
epithelial cells
We used PAR-6 antibodies (Hung and Kemphues, 1999) and GFPtagged PAR-6 (hereafter PAR-6GFP) to examine the localization of
PAR-6 in embryonic epithelial cells. Early embryos contain
maternally supplied PAR-6, which diminishes in level after the onset
of gastrulation (26-cell stage) (Hung and Kemphues, 1999; Nance
and Priess, 2002). Levels of PAR-6 immunostaining increase in
epithelial cells that form during the middle stages of embryogenesis
(~300- to 400-cell stage) (Bossinger et al., 2001; Leung et al., 1999;
McMahon et al., 2001). Epidermal epithelial cells are superficial and
have contact-free apical surfaces that face the eggshell, whereas
pharyngeal and intestinal epithelial cells are internal and have apical
surfaces that contact other epithelial cells and the digestive tract
lumen (Fig. 1A,B). We noted a previously undescribed PAR-6
immunostaining at the apical surfaces of epidermal cells (arrow, Fig.
1A); staining was most intense when epidermal cells first formed
and diminished as embryos aged (compare Fig. 1A,B). PAR-6
antibodies also labeled the apical surfaces of pharyngeal and
intestinal epithelial cells, as has been described by others (Bossinger
et al., 2001; Leung et al., 1999; McMahon et al., 2001), as well as
the excretory cell (Fig. 1B). Because specificity of PAR-6
immunostaining in epithelial cells has not been established, we
analyzed the localization of a functional PAR-6GFP fusion protein
expressed from the par-6 promoter (Nance et al., 2003) (see
Materials and methods). PAR-6GFP was expressed in epidermal,
pharyngeal, intestinal, and excretory epithelial cells, colocalized
with PAR-6 at apical surfaces, and was present even when we
introduced the transgene at fertilization (Fig. 1C,F and data not
shown). In summary, PAR-6 localizes to the apical cortex of most or
all embryonic epithelial cells and at least some PAR-6 in epithelial
cells arises from zygotic par-6 expression.
We performed triple immunostaining experiments to determine
whether PAR-6 colocalized with PKC-3 and PAR-3. In polarizing
epithelial cells, PAR-6 colocalized broadly with both PKC-3 and
PAR-3 (Fig. 1A⬘ and data not shown). In fully polarized epithelial
cells PAR-6 continued to colocalize with PKC-3 at the apical cortex
Fig. 1. PAR-6 localization in epithelial cells. In this and all
subsequent figures, embryos (~50 ␮m in length) or embryo insets are
oriented anterior to the left. Nuclei are stained with DAPI (blue) and
show cell-type differences in staining intensity. (A,B) Lateral views of
embryos when epithelial cells are polarizing (A, bean stage) or after
most epithelial cells have completed polarization (B, 1.5 fold stage).
PAR-6 localizes to apical surfaces of cells in each of the major epithelia
(labeled); staining in epidermal epithelial cells is weaker and more
difficult to detect in older embryos (compare A and B). ␣-PAR-6
antibodies also recognize germline P granules (P). (A⬘,B⬘) Boxed regions
magnified in insets below also show PAR-3 staining; inset contrast has
been adjusted to indicate nuclei. (C-H) Dorsoventral views of polarizing
intestinal cells showing PAR-6GFP and junction proteins. The intestinal
midline is indicated with opposing arrowheads. (C-E) PAR-6GFP and
HMP-1/␣-catenin at initial stages of intestinal cell polarization (before
apical migration of intestinal nuclei). (F-H) PAR-6GFP and DLG-1 in
polarizing intestinal cells (after apical migration of intestinal nuclei).
Scale bars: 2.5 ␮m.
(see Fig. 3B⬘), but PAR-3 developed a more lateral localization (Fig.
1B⬘). Thus, whereas PAR-6 and PKC-3 always appear to colocalize,
PAR-6 and PAR-3 overlap only transiently as epithelial cells
polarize.
To determine when PAR-6 first localizes asymmetrically, we
examined the distribution of PAR-6GFP in intestinal precursor cells
that are just beginning to polarize. Intestinal precursor cells are
arranged in left and right columns; as apicobasal polarity develops,
nuclei migrate to the midline between the columns where the apical
surface forms (compare Fig. 1C,F) (Leung et al., 1999; Sulston et
al., 1983). Prior to apical nuclear movements, we observed spot-like
accumulations of cortical PAR-6GFP at lateral and apical interfaces
of intestinal precursor cells (Fig. 1C). PAR-6GFP puncta congregated
at apical surfaces (Fig. 1F) before organizing into a continuous band
(see Fig. 1B). We co-stained embryos with GFP antibodies and
junction protein antibodies to determine if apical PAR-6GFP
accumulation preceded the formation of apical junctions. The
adherens junction protein HMP-1/␣-catenin and the junction protein
DLG-1/Discs large accumulate in puncta that congregate near the
apical-lateral interface before forming continuous belt-like junctions
(Bossinger et al., 2001; Costa et al., 1998; Firestein and Rongo,
DEVELOPMENT
Embryos were fixed using the freeze-crack methanol procedure and stained
as described (Leung et al., 1999). The following primary antibodies and
dilutions were used: rabbit (Rb) ␣-ACT-5 1:50 (MacQueen et al., 2005),
mouse (Ms) ␣-AJM-1 ‘MH27’ 1:10 (Francis and Waterston, 1991; Koppen
et al., 2001), Rb ␣-EBP-2 1:3000 (Srayko et al., 2005), Ms ‘F2-P3E3⬘ 1:5
(Eisenhut et al., 2005), chicken ␣-GFP 1:200 (Chemicon), Rb ␣-GFP 1:2000
(Abcam), Rb ␣-HMR-1 1:100 (Costa et al., 1998), Ms ␣-HMP-1 1:10 (Costa
et al., 1998), Ms ␣-IFB-2 ‘MH33’ 1:150 (Bossinger et al., 2004; Francis and
Waterston, 1991), Rb ␣-LET-413 1:5000 (Aono et al., 2004), Ms ␣-PAR-3
1:25 (Nance et al., 2003), Rb ␣-PAR-6 1:20 (Hung and Kemphues, 1999),
rat (Rt) ␣-PKC-3 1:30 (Tabuse et al., 1998), Ms ␣-PSD-95 1:200 (Affinity
Bioreagents; recognizes DLG-1) (Firestein and Rongo, 2001), Rt ␣-alphatubulin 1:2000 (Harlan). Primary antibodies were detected using
dye-coupled secondary antibodies (Molecular Probes, Jackson
Immunoresearch). For tubulin plus PAR-6 and EBP-2 plus PKC-3 costainings, secondary antibody incubations and washes were performed
sequentially. Z-stacks of embryos were acquired with a Zeiss Imager, 63⫻
1.4 NA objective and Axiocam MRM camera and were deconvolved using
AxioVision software. Images shown are maximum intensity projections of
several adjacent planes spaced at 300 nm intervals. For embryos stained with
␣-EBP-2 or ␣-tubulin antibodies, confocal images were acquired with a
Zeiss 510 LSM and a 63⫻ 1.3 NA objective. Unless stated otherwise, a
minimum of 50 control and 20 par-6(M/Z) embryos at the indicated stages
were examined in each staining experiment.
Fig. 2. PAR-6ZF1GFP localization and degradation. Panels show par6(zu170) embryos expressing maternal PAR-6ZF1GFP stained with ␣-PAR6 antibodies (green) and/or ␣-GFP antibodies (red). ␣-PAR-6 antibodies
also react non-specifically with P granules (P). (A,B) One-cell embryo costained for PAR-6 and GFP; PAR-6ZF1GFP is at the anterior cortex (arrow).
(C) A 26-cell embryo; PAR-6ZF1GFP has degraded from most cells except
the germ-line precursor cell (asterisk) and the youngest somatic cells
(arrows). (D) ~100-cell embryo; PAR-6ZF1GFP is detectable only in the
two germ cells (asterisks). (E,F) 1.25 fold stage embryos; PAR-6 (arrow
in E) but not PAR-6ZF1GFP (F) is detectable in epithelial cells. Scale bar:
2.5 ␮m.
2001; Koppen et al., 2001; Leung et al., 1999; McMahon et al.,
2001; Segbert et al., 2004) (see Fig. 7). In co-stained embryos, we
observed puncta of PAR-6GFP colocalizing with HMP-1/␣-catenin
even at the earliest stages of polarization (Fig. 1C-E). By contrast,
we observed PAR-6GFP puncta in polarizing intestinal precursor cells
before DLG-1 was expressed; at slightly later stages, many DLG-1
puncta associated loosely with the apical PAR-6GFP domain (Fig. 1FH). Thus, the apical accumulation of PAR-6GFP develops at very
early stages of epithelial polarization, coinciding with or preceding
the apical accumulation of junction proteins.
par-6 maternal-effect-lethal alleles eliminate
maternal but not zygotic PAR-6 expression
The expression and localization of PAR-6 suggested a role in
determining epithelial polarity. However, animals homozygous for
maternal-effect-lethal par-6 alleles (zu170, zu222) do not have
obvious epithelial defects (Watts et al., 1996). Because perduring
maternal PAR-6 might rescue epithelial phenotypes, we designed a
strategy to eliminate maternal PAR-6 from the embryo before
epithelial cells form. We have previously shown that expressing PAR
proteins fused to the ZF1 domain from the germline protein PIE-1
causes the tagged PAR proteins to degrade from somatic cells in the
early embryo (Nance et al., 2003); the normal role of ZF1 is to force
PIE-1 degradation in somatic cells (Reese et al., 2000). Importantly,
maternally expressed ZF1-tagged PAR proteins are present within
the zygote to establish anteroposterior polarity (Nance et al., 2003),
which is required for the subsequent formation of organized
epithelial cells. We constructed a transgene driven by the maternal
Development 134 (7)
pie-1 promoter expressing PAR-6 fused to ZF1 and GFP (hereafter
PAR-6ZF1GFP), then crossed the transgene into a par-6 mutant
background where maternal PAR-6 is not detectably expressed
(Hung and Kemphues, 1999). As we observed with PAR-6ZF1GFP
expressed from the par-6 promoter (Nance et al., 2003), PAR6ZF1GFP expressed from the pie-1 promoter localized to the anterior
cortex of the zygote and to the outer cortex of early embryonic cells
(Fig. 2A-C and data not shown). Unlike par-6 mutant embryos, par6 mutant embryos expressing maternal PAR-6ZF1GFP appeared to
have normal anteroposterior polarity, including an asymmetric first
division and asymmetric localization of germline P granules (‘P’ in
Fig. 2A). PAR-6ZF1GFP levels dropped in somatic cells beginning at
the six- to eight-cell stage (Fig. 2C and data not shown), and after
the 50-cell stage we were unable to detect PAR-6ZF1GFP in any
somatic cell (Fig. 2D and data not shown). In summary, maternal
PAR-6ZF1GFP restores anteroposterior polarity to par-6 mutant
embryos then degrades well before epithelial cells are born.
Surprisingly, most par-6 embryos expressing PAR-6ZF1GFP
appeared to develop normally (803/811 hatched) and grew to fertile
adults. We stained embryos to determine if PAR-6 and PAR-6ZF1GFP
were indeed removed from epithelial cells. As expected, GFP
antibodies did not stain epithelial cells, indicating that maternal
PAR-6ZF1GFP was not present (Fig. 2F). However, epithelial cells in
par-6 embryos expressing maternal PAR-6ZF1GFP showed high levels
of PAR-6 immunostaining; we obtained similar results using two
different par-6 alleles (zu170, zu222) (Fig. 2E and data not shown).
These findings suggest either that PAR-6 immunostaining in
epithelial cells is nonspecific or that par-6 mutations do not prevent
zygotic PAR-6 expression. Because we demonstrate below that
PAR-6 antibody staining is specific, we conclude that par-6(zu170)
and par-6(zu222) prevent maternal but not zygotic PAR-6
expression. Zygotic expression of endogenous PAR-6 could mask
epithelial phenotypes in par-6 mutant embryos expressing PAR6ZF1GFP.
A par-6 deletion mutation causes larval lethality
par-6 alleles defective for maternal but not zygotic PAR-6
expression contain a transposable element insertion in the par-6 3⬘
untranslated region (UTR) (Hung and Kemphues, 1999). We
obtained a par-6 deletion allele (tm1425; provided by the National
Bioresource Project, Tokyo) lacking sequences encoding much of
the N-terminal PB1 domain (green in Fig. 3A), which in mammalian
Par6 is required for binding to aPKC (Lin et al., 2000; Suzuki et al.,
2001). The deletion also causes a frameshift six codons beyond the
deleted region, which is predicted to truncate PAR-6 before the
CRIB and PDZ domains (Fig. 3A and Materials and methods). par6(tm1425) failed to complement existing par-6 alleles and had a
more severe larval lethal phenotype that was rescued by a par-6::gfp
transgene (see Materials and methods). These observations strongly
suggest that par-6(tm1425) inactivates full-length PAR-6 and that
zygotic expression of PAR-6 is required for viability.
Removing maternal and zygotic PAR-6 causes
embryonic lethality and disrupts epithelial cell
adhesion
We performed crosses to obtain par-6(zu170)/par-6(tm1425)
hermaphrodites expressing maternal PAR-6ZF1GFP. Of the selfprogeny from these hermaphrodites, 25% should be par-6(tm1425)
homozygotes lacking both maternal and zygotic PAR-6 and
expressing maternal PAR-6ZF1GFP only at early embryonic stages.
We found that 24.7±1.0% of the embryos arrested before hatching
(n=1304 embryos). To confirm that arrested embryos lacked PAR-6
DEVELOPMENT
1262 RESEARCH ARTICLE
C. elegans PAR-6 and junction formation
RESEARCH ARTICLE 1263
During the second phase of embryogenesis, which is characterized
by morphogenesis, 24% arrested. Two prominent morphogenetic
events occur during this phase of development. At the beginning of
the morphogenesis phase, dorsal epidermal cells migrate ventrally
to encase the embryo in skin (‘enclosure’); subsequently, epidermal
cells undergo cell shape changes that squeeze and elongate the
embryo fourfold in length (‘elongation’; see Fig. 4A) (Chin-Sang
and Chisholm, 2000; Simske and Hardin, 2001). Arrested embryos
completed enclosure, but did not progress beyond the initial stages
of elongation (not beyond the twofold stage) (Fig. 4B,C). Epidermal
cells of arrested embryos eventually began to separate from one
another, allowing internal cells to extrude to the embryo’s surface
(arrowheads in Fig. 4C). Separations were not restricted to the
ventral epidermal cells, which form new contacts during enclosure,
but occurred at many different positions. Because, as demonstrated
above, embryos arresting before the twofold stage lack PAR-6
immunostaining, these timelapse experiments indicate that PAR-6
is required for embryonic elongation and epithelial cell adhesion.
in epithelial cells, we allowed synchronized embryos to develop
through much of embryogenesis then co-stained with PAR-6 and
PKC-3 antibodies. All embryos that developed beyond the twofold
stage (141 of 204, 69%) contained apical PAR-6 and PKC-3 in
epithelial cells (data not shown, see Fig. 3B). All embryos that
arrested before the twofold stage (63 of 204, 31%) lacked PAR-6
immunostaining, and PKC-3 was distributed uniformly throughout
the cytoplasm of epithelial cells (Fig. 3C). As PAR-6 is required to
position PKC-3 asymmetrically in the one-cell embryo and in early
embryonic cells (Nance et al., 2003; Tabuse et al., 1998), these
observations indicate that arrested embryos lack PAR-6 activity in
epithelial cells. We refer to these arrested embryos as ‘par-6(M/Z)’
embryos.
We used 3D timelapse videomicroscopy to determine when par6(M/Z) embryos first developed defects. Using our crossing scheme,
we expected 25% of the embryos analyzed to be par-6(M/Z). All of
the embryos filmed (n=160) appeared to develop normally through
the first half of embryogenesis, when most cell divisions occur.
par-6(M/Z) epithelial cells polarize but develop
abnormal junctions
The cell adhesion defects of par-6(M/Z) embryos suggest that
epithelial cells lacking PAR-6 fail to polarize and/or cannot form
functional apical junctions. We examined epithelial polarity by
staining embryos for cytoskeletal markers that develop apicobasal
asymmetries in intestinal epithelial cells. Microtubules concentrate
at the apical cortex of intestinal epithelial cells as they first polarize
(Leung et al., 1999). Intestinal epithelial cells of wild-type and par6(M/Z) embryos showed similar apical enrichments of both
microtubules and EBP-2/EB1, a plus-end microtubule-binding
protein (Srayko et al., 2005), although in par-6(M/Z) embryos both
apical microtubules and EBP-2 appeared somewhat less compact
than in wild type (Fig. 5A-B⬘ and see Fig. S1A-B⬘ in the
supplementary material). The largely coincident localization of
microtubules and EBP-2 was somewhat surprising, given that the
plus ends of microtubules in mammalian epithelial cells concentrate
basally (Musch, 2004); it is unclear whether these differences reflect
an alternative organization of microtubules in C. elegans epithelial
cells, or whether in C. elegans the microtubules are shorter and thus
present an abundance of plus ends. We also examined the
distribution of the intermediate filament protein IFB-2 and the
microvillar actin ACT-5, the levels of which increase apically at
slightly later stages, as microvilli begin to form at the apical surfaces
of intestinal cells (Bossinger et al., 2004; MacQueen et al., 2005).
When IFB-2 and ACT-5 were first expressed, both proteins showed
apical concentrations in wild-type and par-6(M/Z) intestinal
epithelial cells, although occasional gaps in IFB-2 apical localization
were evident between cells in par-6(M/Z) embryos (see below; Fig.
5C,D and see Fig. S1C,D in the supplementary material). We
Fig. 4. Cell adhesion in par-6(M/Z) embryos. Nomarski micrographs of wild-type and par-6(M/Z) embryos. The wild-type embryo (A) has
elongated threefold; the par-6(M/Z) embryos (B,C) have arrested at the 1.5 fold stage and the embryo in C has developed epidermal lesions
(arrowheads). Scale bar: 2.5 ␮m.
DEVELOPMENT
Fig. 3. The par-6(tm1425) deletion and PAR protein localization in
par-6(M/Z) embryos. (A) The par-6 gene (top) and its predicted fulllength product (bottom). Exons (rectangles), introns (lines), and the
region deleted in tm1425 are indicated. The PB1, CRIB (C) and PDZ
domains are shown in the predicted protein, and sequences encoding
these domains are colored in par-6 exons. (B,C) PAR-6, PKC-3, and PAR3 localization in wild-type (B) and par-6(M/Z) (C) embryos.
(B⬘,C⬘) Magnifications of the boxed regions in B,C. PKC-3 is uniform in
par-6(M/Z) embryos. Nonspecific staining of P granules by PAR-6
antibodies is indicated (P). Scale bars: 2.5 ␮m.
1264 RESEARCH ARTICLE
Development 134 (7)
Fig. 5. Epithelial polarity in par-6(M/Z)
embryos. (A-B⬘) Microtubules in wild-type
(A,A⬘) and par-6(M/Z) (B,B⬘) embryos
concentrate at apical regions of intestinal
epithelial cells (arrows in A⬘,B⬘); embryos are costained for ␣-tubulin and PAR-6. (C-E) IFB-2
localization in intestinal cells of wild-type and
par-6(M/Z) embryos. IFB-2 localizes in a
continuous apical band in wild type (C). In
younger par-6(M/Z) embryos (D), IFB-2 is apical
but not always continuous between cells
(arrow); (E) apical IFB-2 patches separate and
circularize (arrow) in older par-6(M/Z) embryos.
Scale bars: 2.5 ␮m.
Rongo, 2001; Koppen et al., 2001; McMahon et al., 2001), we
created ‘double mutants’ to ask whether PAR-6 functions
redundantly with HMR-1 or DLG-1 to polarize epithelial cells.
Using RNAi to deplete embryos of DLG-1 or a combination of
RNAi and the hmr-1(zu248) mutation to deplete embryos of HMR1, we were unable to detect either protein in intestinal epithelial cells
(see Materials and methods). Consistent with reports by others
(Bossinger et al., 2001; Costa et al., 1998; Firestein and Rongo,
2001; Koppen et al., 2001; McMahon et al., 2001), we observed that
DLG-1 and PKC-3 localized apically in intestinal cells of embryos
depleted of HMR-1 (n=27/28), and HMP-1 and PAR-6 were apical
in intestinal cells of embryos depleted of DLG-1 (n=118) (see Fig.
S2C,E in the supplementary material). Depleting HMR-1 (stained
with DLG-1, n=36) or DLG-1 (stained with HMP-1, n=40) in par6(M/Z) embryos yielded phenotypes similar to those of par-6(M/Z)
embryos: in each case junction proteins were apical but fragmented
(see Fig. S2D,F in the supplementary material). In contrast to HMR1 and DLG-1, the basolateral protein LET-413/Scribble is required
for formation of continuous junctions and to maintain the apical
localization of junction proteins and other apical proteins (Bossinger
et al., 2001; Koppen et al., 2001; Legouis et al., 2000; McMahon et
al., 2001). Similar to observations by others (Bossinger et al., 2001;
Koppen et al., 2001; Legouis et al., 2000; McMahon et al., 2001),
we noted that PAR-6 and DLG-1 spread laterally in intestinal cells
of let-413(RNAi) embryos and junctions appeared fragmented
(87/89 embryos). let-413(RNAi) par-6(M/Z) embryos stained for
DLG-1 showed an indistinguishable phenotype (24/24 embryos). In
summary, our experiments do not provide evidence for PAR-6
functioning redundantly with HMR-1, DLG-1 or LET-413 to
polarize intestinal epithelial cells. However, these experiments do
not address whether PAR-6 regulates epithelial polarity through
more complex redundant interactions.
PAR-6 is required to condense apical junction
proteins
In wild-type embryos, puncta of junction proteins such as DLG-1
congregate at the apicolateral surface and condense into belt-like
apical junctions (Bossinger et al., 2001; Koppen et al., 2001;
McMahon et al., 2001). The abnormal organization of junction
proteins in par-6(M/Z) embryos could result from a failure to
initially condense junction proteins or a failure to prevent continuous
junctions from fragmenting during the stresses of morphogenesis.
To determine when PAR-6 function is first required during apical
junction formation, we performed timelapse fluorescence
microscopy experiments to follow the movements of GFP-tagged
DLG-1 (DLG-1GFP) in par-6(M/Z) and control embryos (Fig. 7 and
see Movies 1, 2 in the supplementary material). We imaged junction
DEVELOPMENT
observed apical localization of IFB-2 in par-6(M/Z) embryos when
using a different transgene insertion (zuIs44) to express maternal
PAR-6ZF1GFP, either of two maternal alleles of par-6 (zu170, zu222)
in our crossing scheme, or after treating worms to induce RNAi of
any hypothetical remaining endogenous PAR-6 (n=11 par-6(M/Z)
embryos; see Materials and methods). In summary, the asymmetric
localization of microtubules, IFB-2 and ACT-5 in par-6(M/Z)
embryos indicates that detectable levels of PAR-6 and asymmetric
localization of PKC-3 are not needed for C. elegans embryonic
epithelial cells to polarize.
In older arrested par-6(M/Z) embryos, apical IFB-2 and ACT-5
were often present in isolated apical patches (Fig. 5E and data not
shown). These apical patches did not appear to result from apoptosis
of epithelial cells as par-6(M/Z) embryos showed no increase in
staining with the F2-P3E3 antibody (data not shown), which
recognizes apoptotic bodies (Eisenhut et al., 2005). Rather, we
interpret these isolated apical patches as the result of failed adhesion
between adjacent epithelial cells. Together with our 3D timelapse
analysis, these staining experiments suggest that epithelial cells in
par-6(M/Z) embryos develop apicobasal polarity but eventually
detach from one another.
To determine whether abnormal apical junctions might explain
the adhesion defects we observed, we examined the localization of
junction proteins HMR-1/E-cadherin, HMP-1/␣-catenin, DLG1/Discs large, and AJM-1 in par-6(M/Z) embryos. Each of these
junction proteins was positioned apically within intestinal cells;
however, in contrast to the continuous belt-like organization of
HMP-1, HMR-1, DLG-1, and AJM-1 in wild-type embryos, each
protein showed a fragmented distribution (Fig. 6 and data not
shown). In some cell types, especially lateral epidermal cells, HMP1 and HMR-1 fragmentation was often less severe than DLG-1 or
AJM-1 fragmentation. PAR-3 showed a similar apical but
fragmented localization in par-6(M/Z) embryos (Fig. 3C). These
defects in apical junction protein and PAR-3 organization were not
caused by mislocalization of the basolateral protein LET-413 (Fig.
6E,F), which is also required to form continuous apical junctions
(Legouis et al., 2000). In summary, apical junction proteins, PAR-3,
and the basolateral protein LET-413 can become positioned
asymmetrically without PAR-6, but apical junction proteins and
PAR-3 require PAR-6 for their organization into belt-like structures.
Our failure to observe epithelial polarity defects in par-6(M/Z)
embryos could be explained if PAR-6 and another protein(s)
function redundantly to polarize epithelial cells. Because E-cadherin
and Discs large are required to polarize Drosophila epithelial cells
(Humbert et al., 2003) but their C. elegans orthologs (HMR-1/Ecadherin and DLG-1/Discs large) are dispensable for epithelial
polarity (Bossinger et al., 2001; Costa et al., 1998; Firestein and
Fig. 6. Apical junctions in par-6(M/Z) embryos. Wild-type or par6(M/Z) embryos immunostained as indicated. Dashed boxes are
magnified in the insets below; the contrast within insets has been
increased to highlight intestinal epithelial cell staining. Nonspecific
staining of P granules by PAR-6 antiserum is indicated (P). (A,B) PAR-6
and HMP-1. In wild-type (A), PAR-6 is apical and HMP-1 is present in
more lateral junctions. HMP-1 is apical but fragmented in par-6(M/Z)
embryos (B). (C,D) HMR-1 and DLG-1. In wild type (C), HMR-1 and
DLG-1 are present together in apical junctions within adjacent domains.
In par-6(M/Z) embryos (D), HMR-1 and HMP-1 are apical but
fragmented and do not colocalize (arrow, D⬘). (E,F) LET-413 and PAR-3
in epidermal cells, lateral view in bean stage embryos. In both wild-type
(E) and par-6(M/Z) (F) embryos, LET-413 is found at lateral surfaces
(arrows) but not apical surfaces (arrowheads); PAR-3 is present only at
the apicolateral interface. Scale bars: 2.5 ␮m.
formation in epidermal cells, which are superficial and have apical
surfaces easily observed within the same focal plane. As others have
described (Bossinger et al., 2001; Koppen et al., 2001; McMahon et
al., 2001), we noted that puncta of DLG-1GFP in control embryos
coalesced rapidly into continuous belt-like junctions (16/16
embryos). DLG-1GFP in par-6(M/Z) embryos was expressed at a
similar stage and formed apical puncta, but these puncta failed to
condense into continuous junctions (9/9 embryos). In addition, we
never observed continuous DLG-1 in intestinal epithelial cells of
par-6(M/Z) embryos at stages when DLG-1 becomes continuous in
wild type (Fig. 7C,D). We conclude that PAR-6 is required for the
initial formation of continuous apical junctions.
DISCUSSION
PAR-6 functions reiteratively to control cell
adhesion
Our results show that PAR-6 is essential for epithelial cells to remain
adherent during elongation. This is not the first stage of
embryogenesis in which PAR-6 is needed for proper cell-cell
RESEARCH ARTICLE 1265
Fig. 7. DLG-1 localization during junction formation.
(A,B) Fluorescence timelapse movie stills (taken from Movies 1, 2 in the
supplementary material) of wild-type (A) and par-6(M/Z) (B) epidermal
cells expressing DLG-1GFP. Times are indicated in minutes:seconds, with
00:00 representing the time DLG-1GFP is first expressed in epidermal
cells. DLG-1GFP forms continuous junctions in wild-type but not par6(M/Z) embryos. (C,D) DLG-1 and PKC-3 localization in intestinal
epithelial cells of wild-type (C) and par-6(M/Z) (D) embryos. Embryos are
at the 1.25 fold stage, just after DLG-1 organizes into continuous
junctions in wild-type embryos. DLG-1 in par-6(M/Z) embryos is always
fragmented. Scale bars: 2.5 ␮m.
adhesion – early embryonic cells lacking PAR-6 can separate from
one another inappropriately. The different surfaces of early
embryonic cells show contrasting patterns of adhesion with
neighboring cells. For example, the lateral surfaces of cells (which
contact adjacent cells) are very adherent, whereas the inner surfaces
of cells (which face the interior of the embryo) separate from one
another to form a small centrally positioned blastocoel cavity (Nance
and Priess, 2002). PAR-6 localizes to the outer surfaces of early
DEVELOPMENT
C. elegans PAR-6 and junction formation
embryonic cells (Hung and Kemphues, 1999; Nance and Priess,
2002) and is required to prevent lateral surfaces from forming
blastocoel-like separations (Nance et al., 2003).
Despite these analogies, the mechanism by which PAR-6
regulates adhesion in early embryonic cells and epithelial cells
probably differs. Adhesion between epithelial cells is mediated in
part by apical junctions. For example, removing both DLG-1 and
HMP-1/␣-catenin causes epithelial cells to separate from one
another (Segbert et al., 2004). Because in par-6(M/Z) embryos
apical DLG-1 and HMP-1 are fragmented rather than belt-like, it
is likely that the adhesion defects we observed are caused by
defective junctions. By contrast, early embryonic cells do not form
apical junctions (Costa et al., 1998; Nance and Priess, 2002).
Adherens junction proteins such as HMR-1/E-cadherin and HMP1/␣-catenin are instead found at all sites of cell-cell contact, are not
needed for cell adhesion, and do not require PAR-6 to localize
(Costa et al., 1998; Nance et al., 2003) (our unpublished
observations). PAR-6 regulates early embryonic cell adhesion and
epithelial cell adhesion independently, as embryos lacking PAR-6
in early embryonic cells but expressing PAR-6 in epithelial cells
do not develop fragmented junctions or epithelial cell adhesion
defects (Nance et al., 2003).
Continuous apical junctions never form in par-6(M/Z) embryos,
so why do epithelial cells only show adhesion defects at later stages
after elongation has begun? One possibility is that apical junctions
become more defective over time. Although this is difficult to
exclude, we observed that the fragmented organization of DLG-1GFP
in par-6(M/Z) epithelial cells does not worsen in timelapse movies
as long as 2 hours (our unpublished observations). Instead, we
propose that junctions are not needed to hold cells together during
the sheet-like epithelial cell movements of epidermal enclosure, but
are required to resist tension known to develop during elongation
(Priess and Hirsh, 1986). Studies of morphogenetic movements in
other organisms indicate that epithelial cells do not always require
intact junctions to adhere or migrate. For example, sheet-like
movements of zebrafish myocardial precursor cells can still occur
when junctions are disrupted by mutation of the pkc-3 homolog
heart and soul (prkci) (Rohr et al., 2005).
PAR-6, PKC-3 and epithelial polarity
Our results show that full-length PAR-6 and asymmetrically
localized PKC-3 are not needed for embryonic epithelial cells to
polarize. For example, we observed that apical cytoskeletal markers
(microtubules, ACT-5, IFB-2), apical junction proteins (PAR-3,
HMR-1, HMP-1, DLG-1 and AJM-1), and the basolateral protein
LET-413 all localize asymmetrically in par-6(M/Z) embryos. In
principle, the ability of par-6(M/Z) epithelial cells to polarize could
be explained by incomplete removal of PAR-6 or PAR-6ZF1GFP.
Several observations suggest that this is very unlikely. First, we were
unable to detect PAR-6 or PAR-6ZF1GFP in epithelial cells of par6(M/Z) embryos. Second, phenotypes of par-6(M/Z) embryos did
not become stronger when we used RNAi to knockdown any
hypothetical remaining PAR-6. Third and most convincing, PKC-3
never localized apically in par-6(M/Z) epithelial cells; in Drosophila
epithelial cells and C. elegans early embryonic cells, aPKC/PKC-3
apical positioning requires PAR-6 (Hutterer et al., 2004; Nance et
al., 2003). As par-6(tm1425) does not remove the complete par-6
coding sequence, we cannot exclude the possibility that par-6(M/Z)
embryos express alternative isoforms of PAR-6 not detected by
PAR-6 antibodies. However, as tm1425 deletes much of the PKC-3binding (PB1) domain, any alternative isoforms would not likely
bind PKC-3.
Development 134 (7)
The epithelial phenotype of C. elegans par-6(M/Z) embryos
contrasts with that of Drosophila blastoderm epithelial cells lacking
Par-6. Blastoderm epithelial cells begin to polarize as they form by
cellularization, when membranes invaginate to separate nuclei in the
syncytial embryo (Lecuit, 2004). In embryos lacking both maternal
and zygotic Par-6, the apical protein Patj, the apical junction protein
Arm/␤-catenin, and Baz/PAR-3 are all mislocalized to lateral surfaces
(Hutterer et al., 2004; Petronczki and Knoblich, 2001). The different
phenotypes of fly and worm epithelial cells lacking PAR-6 might
reflect an evolving role for PAR-6 in epithelial polarity. Alternatively
or in addition, PAR-6 might be utilized differently in epithelial cells
that form by cellularization (Drosophila blastoderm cells) versus
epithelial cells that differentiate from groups of nonpolarized
precursors (C. elegans embryonic cells). Cultured mammalian
epithelial cells such as MDCK cells also form from nonpolarized
precursor cells, and Par6 regulates tight junction formation in these
cells (see Introduction). However, because cell lines lacking all Par6
activity have not been described, it is not yet known if mammalian
Par6 also controls other epithelial asymmetries.
If PAR-6 is not required to polarize C. elegans epithelial cells,
which pathways are utilized? One possibility is that C. elegans
epithelial cells rely on a combination of polarization pathways, and
inactivation of any single pathway might not fully block polarization.
In Drosophila, PAR proteins function coordinately with other
pathways to regulate epithelial polarity (Humbert et al., 2003; Macara,
2004). For example, adherens junction proteins such as E-cadherin,
basolateral proteins such as Discs large and Scribble, and apical
proteins such as Crumbs function in different pathways that each
contribute to epithelial polarization. Homologues of these proteins are
not essential for the initial polarization of epithelial cells in C. elegans
(Bossinger et al., 2001; Costa et al., 1998; Firestein and Rongo, 2001;
Koppen et al., 2001; McMahon et al., 2001), although LET413/Scribble is required for proper positioning and compaction of
junction proteins and for preventing the progressive lateral spread of
apical proteins such as PAR-6 (Bossinger et al., 2001; Koppen et al.,
2001; Legouis et al., 2000; McMahon et al., 2001). Our double-mutant
experiments suggest that simple redundant interactions between PAR6 and HMR-1, DLG-1, or LET-413 are not required for epithelial cells
to polarize. Additionally, in RNAi experiments we failed to detect
redundant interactions between PAR-6 and CRB-1/Crumbs (our
unpublished observations), although we could not verify depletion of
CRB-1 because CRB-1 antiserum is no longer available (O.
Bossinger, personal communication). Despite these observations, it is
possible that complex redundant interactions between combinations
of these pathways mediate epithelial polarization.
Alternatively, epithelial polarity could be mediated by PAR-3,
independently of PAR-6 and PKC-3. Indeed, Baz/PAR-3 acts
upstream of aPKC to polarize Drosophila blastoderm epithelial cells
and is also required in MDCK cells for tight junction formation
(Chen and Macara, 2005; Harris and Peifer, 2005). C. elegans PAR3 is required for the apical localization of AJM-1 and microfilaments
in epithelial cells of the distal spermatheca (Aono et al., 2004),
suggesting that it might be essential for polarity in all epithelial cells.
Interestingly, Drosophila Baz/PAR-3 localizes to a different apical
domain than Par-6 or aPKC/PKC-3 (Harris and Peifer, 2005), and
we noted a similar spatial organization of PAR-3, PAR-6, and PKC3 in C. elegans embryonic epithelial cells.
PAR-6 and apical junctions
PAR-6 appears to have at least partly different functions than LET413 or DLG-1 in regulating junction biogenesis. Whereas LET-413
is required to properly compact junction proteins such as HMP-1
DEVELOPMENT
1266 RESEARCH ARTICLE
and to prevent their lateral spread (Koppen et al., 2001; Legouis et
al., 2000; McMahon et al., 2001), DLG-1 plays only a minor role in
compacting HMP-1 and is not needed for HMP-1 apical localization
(Bossinger et al., 2001; Firestein and Rongo, 2001; Koppen et al.,
2001; McMahon et al., 2001). par-6(M/Z) embryos have a
phenotype distinct from that of let-413 mutant embryos and dlg1(RNAi) embryos. For example, both PAR-6 and LET-413 are
required for the integrity of apical junctions, but apical proteins do
not spread laterally in par-6(M/Z) mutant embryos as they do in let413 mutant embryos. Additionally, PAR-6 and DLG-1 are each
required for proper compaction of HMP-1 in apical junctions, but
HMP-1 fragmentation is more severe in par-6(M/Z) mutant embryos
than that reported for dlg-1(RNAi) embryos (Bossinger et al., 2001;
Firestein and Rongo, 2001; Koppen et al., 2001; McMahon et al.,
2001). These observations suggest that PAR-6, LET-413 and DLG1 regulate, at least partially, different aspects of junction biogenesis
and maintenance. Because we found that junction proteins show an
equivalent lateral spread in let-413(RNAi) embryos and let413(RNAi) par-6(M/Z) double-mutant embryos, the ectopic
junctions observed in embryos lacking LET-413 are not caused by
ectopic PAR-6 activity at the lateral cortex.
Our imaging experiments on DLG-1GFP in par-6(M/Z) embryos
indicate that PAR-6 is required for the initial condensation of apical
junction proteins into belt-like structures. The localization and
timing of expression of PAR-6 is consistent with such a role. For
example, we observed that levels of PAR-6GFP increase at apical
surfaces earlier than DLG-1 and AJM-1 and at the same time as
HMP-1 and HMR-1. During the early stages of polarization, lateral
and apical spots of PAR-6GFP showed extensive colocalization with
spots of HMR-1 and HMP-1, suggesting that they may be targeted
to the apical cortex together via a common mechanism.
Selective inhibition of maternal expression by
par-6 maternal-effect-lethal mutations
Original alleles of par-6 were identified in maternal-effect lethal
screens requiring that homozygous mutant animals survive to
adulthood (Watts et al., 1996). Our experiments demonstrate that
these par-6 alleles prevent maternal but not zygotic expression of
PAR-6. Both zu170 and zu222 are insertions of the Tc1 transposable
element into the par-6 3⬘ UTR and do not alter par-6 coding
sequences (Hung and Kemphues, 1999). A Tc1 insertion within
sequences 3⬘ of the pop-1 stop codon also prevents maternal but not
zygotic expression of POP-1 (Lin et al., 1995). Tc1 elements are
targets of RNAi within the germline (Sijen and Plasterk, 2003),
suggesting that maternal mRNAs containing Tc1 elements may be
inactivated by RNAi. Thus introducing Tc1 sequences into the UTR
of a gene might be a general strategy to selectively eliminate its
maternal expression.
We thank Greg Hermann, Ken Kemphues, Renaud Legouis, Shohei Mitani and
the National Bioresource Project, Chris Rongo and Jim Waddle for generously
providing antibodies and strains, and James Priess for helping to create the
zuIs43 and zuIs44 insertions. Thanks also to Scott Clark and Jessica Treisman
for comments on the manuscript. The MH27 and MH33 monoclonal
antibodies were obtained from the Developmental Studies Hybridoma Bank
developed under the auspices of the NICHD and maintained by The University
of Iowa. Some nematode strains used in this work were provided by the
Caenorhabditis Genetics Center, which is funded by the NIH National Center
for Research Resources (NCRR). Funding for this study was provided by an
American Heart Association Heritage Affiliate Grant-in-Aid 0655893T (J.N.)
and a Fulbright Cyprus-America Scholarship (A.A.).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/7/1259/DC1
RESEARCH ARTICLE 1267
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1268 RESEARCH ARTICLE